Sialoadhesin-related compositions and methods

ABSTRACT

Methods of delivering a cargo moiety to a cell is provided that include contacting a cell expressing sialoadhesin with a conjugate including a sialoadhesin binding moiety and a cargo moiety. The sialoadhesin binding moiety binds to the sialoadhesin expressed by the cell and is internalized along with the cargo, delivering the cargo moiety to the cell. Particular methods include induction or enhancement of sialoadhesin expression in a cell which naturally produces little or no sialoadhesin. Induction or enhancement of sialoadhesin expression includes transfection of a sialoadhesin expression construct and/or administration of an agent effective to induce or enhance sialoadhesin expression. Methods and compositions for stimulating an immune response in a subject are detailed. Particular methods and compositions for stimulating an immune response to a virus are provided herein.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation in part of co-pending U.S. Ser. No.13/066,341, filed on Apr. 11, 2011, which is a divisional of co-pendingU.S. patent application Ser. No. 12/227,106, filed Nov. 7, 2008, nowU.S. Pat. No. 7,998,485 (issued Aug. 16, 2011), which is a nationalphase entry under 35 U.S.C. §371 of International Patent ApplicationPCT/IB2007/004499, filed May 11, 2007, published in English asInternational Patent Publication WO 2008/093166 A2 on Aug. 7, 2008,which application claims the benefit under 35 U.S.C. §119(e) and underArticle 8 of the PCT to U.S. Provisional Patent Application Ser. No.60/799,566, filed May 11, 2006, the entire contents of each of which areincorporated herein by reference.

STATEMENT ACCORDING TO 37 C.F.R. §1.821(c) or (e)—SEQUENCE LISTINGSUBMITTED AS A TXT AND PDF FILES

Pursuant to 37 C.F.R. §1.821(c) or (e), files containing a TXT versionand a PDF version of the Sequence Listing have been submittedconcomitant with this application, the contents of which are herebyincorporated by reference.

TECHNICAL FIELD

The disclosure relates generally to biotechnology, and more particularlyto compositions and methods for targeted cargo delivery to a cell. Inparticular, the disclosure relates to compositions and methods fortargeted cargo delivery to a sialoadhesin-expressing cell.

BACKGROUND

Specific delivery of a substance to a targeted cell is desirable forvarious purposes, including pharmacological intervention as well asclinical and research bioassays.

Targeted delivery is particularly desirable where exposure ofnon-targeted cells to a substance to be delivered is preferably avoided,such as where exposure of non-targeted cells can result in undesirableside effects. For example, a therapeutic intervention may requireelimination, inhibition, stimulation and/or activation of a particularcell or cell type. In such a situation, it is advantageous to deliver asubstance effective to achieve a desired result preferentially to atargeted cell in order to avoid an undesirable side effect such asinhibition or stimulation of non-targeted cells and cell types. Targeteddelivery also allows use of less of the substance to be delivered toachieve a desired effect.

SUMMARY OF THE DISCLOSURE

Provided are methods of delivering a cargo moiety to a cell thatincludes contacting a cell expressing sialoadhesin with a conjugateincluding a sialoadhesin binding moiety and a cargo moiety. Thesialoadhesin binding moiety binds to the sialoadhesin expressed by thecell and is internalized along with the cargo moiety, thereby deliveringthe cargo moiety to the cell. Cells naturally expressing sialoadhesinare known, particularly including macrophages. Particular methodsprovided herein include induction or enhancement of sialoadhesinexpression in a cell which naturally produces little or no sialoadhesin.Induction or enhancement of sialoadhesin expression includestransfection of a sialoadhesin expression construct and/oradministration of an agent effective to induce or enhance sialoadhesinexpression. Expression of sialoadhesin is determined by any of variousmethods including binding of sialoadhesin-specific antibodies, detectionof sialoadhesin encoding mRNA and the like.

In certain embodiments, the sialoadhesin binding moiety is an antibodythat binds substantially specifically to sialoadhesin. Such antibodiesinclude, but are not limited to, mouse anti-porcine sialoadhesin mAb41D3, mouse anti-human sialoadhesin mAb 7D2, and mouse anti-porcinesialoadhesin mAb MCA2316.

In further embodiments, a sialoadhesin binding moiety is a sialoadhesinligand.

A cargo moiety included in a conjugate is a stimulator of a response inthe cell in particular embodiments. For example, in certain embodiments,a conjugate is a stimulator of an immune response in the cell. Infurther embodiments, a conjugate which stimulates an immune response inthe cell stimulates an immune response in a subject. Thus, a cargomoiety may be an antigen.

Also provided are embodiments hereof in which the cargo moiety is aninhibitor of the cell. For example, a cargo moiety included in aconjugate is a cytotoxic agent in particular embodiments. A cytotoxicagent is exemplified by, but not limited to, a ribosome inactivatingprotein. A specific cytotoxic agent which is a ribosome inactivatingprotein is saporin.

In further embodiments, a cargo moiety is an antimicrobial agent. Anantimicrobial agent included in a conjugate is effective to inhibit amicrobe such as, but not limited to, a bacterium, a virus, a fungus or aprotozoan.

A cytokine is a cargo moiety in certain embodiments of the method.Optionally, the cargo moiety is a nucleic acid. A delivered nucleic acidmay be an expression construct. Further optionally an expressionconstruct is included in a vector, such as, but not limited to, abacterial plasmid or a viral vector. A nucleic acid cargo may be anantisense construct such as, but not limited to, an antisenseoligonucleotide, an siRNA, an shRNA or an expression vector forexpressing an antisense nucleic acid.

Where a cargo moiety is an antigen, the antigen may be a protein, apeptide, a glycoprotein or a glycopeptide. Such antigens may besynthetic, such as, but not limited to, recombinantly produced orchemically synthesized proteins or peptides; or natural, such as, butnot limited to, an antigen isolated from a cell, virus or organism. Inparticular embodiments, a cargo which is an antigen is a viral protein,a viral peptide, a viral glycoprotein or a viral glycopeptide.

An antigen conjugated to a sialoadhesin binding moiety may be aninfluenza virus hemagglutinin or an antigenic portion thereof. Aspecific antigenic portion of an influenza virus hemagglutinin isencoded by SEQ ID NO:3 (of the accompanying and incorporated hereinSEQUENCE LISTING) or a homologue thereof. In particular embodiments, avirus hemagglutinin included in a conjugate hereof is an influenza virushemagglutinin of SEQ ID NO:4 or a homologue thereof.

A cell contacted by a conjugate for delivery of a cargo to the cell isin vitro, or in vivo.

In further embodiments, a cell is treated with a cytokine effective toinduce or enhance expression of sialoadhesin in the cell. For example, acell treated with a cytokine effective to induce or enhance expressionof sialoadhesin is a monocyte, a monocyte cell line, a macrophage and amacrophage cell line. In particular embodiments, a human cell and/or ahuman-derived cell line is treated with a cytokine effective to induceor enhance expression of sialoadhesin. An example of a human-derivedcell line is human monocyte cell line THP-1. In further embodiments, aporcine cell and/or a porcine-derived cell line is treated with acytokine effective to induce or enhance expression of sialoadhesin.Suitable cytokines effective to induce or enhance expression ofsialoadhesin include interferon alpha (IFN-alpha), and a combination oftumor necrosis factor-alpha (TNF-alpha) and interferon-gamma(IFN-gamma).

Compositions are provided herein that include a sialoadhesin bindingmoiety conjugated to a cargo moiety. The sialoadhesin binding moiety isan antibody or a sialoadhesin ligand in particular embodiments of acomposition hereof.

A method of stimulating an immune response in a subject to a viralantigen is provided herein which includes administering a compositionincluding a sialoadhesin binding moiety conjugated to a viral antigen toa subject. In particular embodiments, a cargo which is a viral antigenis a viral protein, a viral peptide, a viral glycoprotein or a viralglycopeptide.

In particular embodiments, a viral antigen conjugated to a sialoadhesinbinding moiety is an influenza virus hemagglutinin or an antigenicportion thereof. A specific antigenic portion of an influenza virushemagglutinin is encoded by SEQ ID NO:3.

Also provided are methods and compositions in which a viral antigenconjugated to a sialoadhesin binding moiety is a Porcine Reproductiveand Respiratory Syndrome virus (PRRSV), a PRRSV protein or an antigenicportion of a PRRSV protein.

Optionally, a sialoadhesin binding moiety is an antibody or asialoadhesin ligand. Specific antibodies included in a conjugate hereininclude monoclonal antibody 41D3, monoclonal antibody 7D2 and monoclonalantibody MCA2316.

A conjugate including a sialoadhesin binding moiety and a cargo isproduced by chemical bonding between the sialoadhesin binding moiety andcargo in particular embodiments. In further embodiments, a conjugateincluding a sialoadhesin binding moiety and a cargo is produced byrecombinant techniques, including expression of a fusion protein.

In particular embodiments, a method of stimulating an immune responseherein includes administering an amount of a cytokine effective toinduce or enhance expression of sialoadhesin in a cytokine responsivecell in the subject. A specific cytokine effective to induce or enhanceexpression of sialoadhesin in an INF-alpha responsive cell is INF-alpha.A, INF-alpha responsive cell is identified by methods known in the artincluding, but not limited to, detection of an INF-alpha receptor.Particular INF-alpha responsive cells include monocytes, such as, butnot limited to, human monocytes, and monocyte-derived cell lines, suchas, but not limited to, human monocyte cell line THP-1. A macrophage isa further example of an INF-alpha responsive cell.

A method of stimulating an immune response to an antigen in a subject isprovided according to embodiments hereof which includes administering acomposition including a sialoadhesin binding moiety conjugated to anantigen to a subject.

In further embodiments hereof, a method of screening a compound forsialoadhesin binding activity and/or sialoadhesin binding stimulatedcell internalization activity is provided which includes administering acytokine effective to induce or enhance sialoadhesin expression;administering the compound; and performing an assay for specific bindingof the compound to sialoadhesin and/or performing an assay forsialoadhesin binding stimulated cell internalization activity. Inparticular embodiments, the cytokine effective to induce or enhancesialoadhesin expression is INF-alpha. A compound is illustratively ananti-sialoadhesin antibody or a sialoadhesin ligand. Examples of assaysto determine specific binding of the compound include incubation of thecompound with the cell under typical sialoadhesin binding moiety bindingconditions, such as under substantially physiological conditions, anddetection of binding. Detection of binding may include, for instance,detection of a reporter bound to the compound. Detection ofinternalization of the compound is illustratively accomplished bypermeabilization of a cell and incubation with a reagent that binds tothe compound, such as, but not limited to, an antibody, followed bydetection of the reagent.

A method of transfecting a cell is provided which includes administeringa sialoadhesin binding moiety conjugated to an expression construct to acell expressing sialoadhesin. The cell expressing sialoadhesin is a celltransfected with a sialoadhesin expression construct in particularembodiments. In further embodiments, the cell is a cell line stablyexpressing sialoadhesin. In yet further embodiments, the cell is treatedwith a cytokine to induce or enhance sialoadhesin expression.

A kit for delivering a cargo to a cell is provided herein which includesa cell expressing sialoadhesin and a sialoadhesin binding moiety.Optionally, such a kit includes a reagent for use in conjugation of acargo to the sialoadhesin binding moiety. A cell included in such a kitmay be a cell line. In a further option, the sialoadhesin binding moietyincluded in the kit is conjugated to a cargo. The cargo may beconjugated to the sialoadhesin binding moiety is an expressionconstruct.

A method of treating a pathological condition in a subject is providedincluding administering a therapeutically effective amount of asialoadhesin binding moiety conjugated to a therapeutic cargo moiety tothe subject, wherein the therapeutic cargo moiety is delivered to asialoadhesin expressing cell in the subject, thereby treating thepathological condition. In particular embodiments, the therapeutic cargomoiety is an inhibitor of the cell, such as, but not limited to, acytotoxic agent. An example of an inhibitor is saporin. A pathologicalcondition treated according to the method is characterized by presenceof a pathogen in the cell in particular embodiments. In furtherembodiments, the pathological condition is an autoimmune disease orcancer. An included cargo moiety is an inhibitor of macrophageactivation and/or inflammation in embodiments for treating autoimmunedisease. Such inhibitors include, but are not limited to, an inhibitorof macrophage activation and/or inflammation such as IL-10, TGF-beta,6-(methylsulfinyl)hexyl isothiocyanate, a sesquiterpene chromone, and acombination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph illustrating specific binding and internalization of asialoadhesin binding moiety at different times after incubation ofmacrophages at 37° C. with mAb 41D3;

FIG. 2A is a graph showing the mean±SEM of antigen-specific IgM serumtiters after primary immunization with a sialoadhesin bindingmoiety/antigen conjugate herein in which square symbols indicate pigsimmunized with HSA coupled to Sn-specific mAb 41D3; triangle symbolsindicate pigs immunized with HSA coupled to irrelevant control mAb; andcircle symbols indicate pigs immunized with free HSA;

FIG. 2B is a graph showing the mean±SEM of antigen-specific IgG serumtiters after primary immunization with a sialoadhesin bindingmoiety/antigen conjugate herein in which square symbols indicate pigsimmunized with HSA coupled to Sn-specific mAb 41D3; triangle symbolsindicate pigs immunized with HSA coupled to irrelevant control mAb; andcircle symbols indicate pigs immunized with free HSA;

FIG. 2C is a graph illustrating means±SEM of antigen-specific IgG serumtiters after booster immunization in which square symbols indicate pigsimmunized with HSA coupled to Sn-specific mAb 41D3; triangle symbolsindicate pigs immunized with HSA coupled to irrelevant control mAb; andcircle symbols indicate pigs immunized with free HSA;

FIG. 3 is a graph illustrating the percentage of living cells in apopulation of sialoadhesin expressing cells treated with sialoadhesinbinding moiety/cytotoxic agent conjugate compared to cells treated witha non-sialoadhesin binding moiety/cytotoxic agent conjugate;

FIG. 4 is a graph illustrating the percentage of living cells in apopulation of sialoadhesin expressing cells treated with sialoadhesinbinding moiety/cytotoxic agent conjugate compared to non-sialoadhesinexpressing cells treated with a sialoadhesin binding moiety/cytotoxicagent conjugate;

FIG. 5 is a set of histograms generated from flow cytometric analysisshowing binding and internalization of a particular sialoadhesin bindingmoiety;

FIG. 6A is a xerographic reproduction of a digital image showingSDS-PAGE analysis of the presence and purity of native influenzavirushemagglutinin in different fractions obtained during purificationincludes detection of HA via western blotting using a monoclonalantibody directed against HA of the HIN1 virus;

FIG. 6B is a xerographic reproduction of a digital image showingdetection of total protein in the samples shown in FIG. 6A usingCoomassie blue staining;

FIG. 7A is a xerographic reproduction of a digital image showingSDS-PAGE analysis of the production of recombinant HA with a V5-His tagwhere the protein is produced in the absence of fetal bovine serum;

FIG. 7B is a xerographic reproduction of a digital image showingSDS-PAGE analysis of the production of recombinant HA with a V5-His tagwhere the protein is produced in the presence of fetal bovine serum;

FIG. 8A is a xerographic reproduction of a digital image showingSDS-PAGE Western blot analysis of different fractions taken during thepurification process of HA;

FIG. 8B is a xerographic reproduction of a digital image showingSDS-PAGE Coomassie blue analysis of the same fractions taken during thepurification process of HA shown in FIG. 8A;

FIG. 9A is a xerographic reproduction of a digital image showingSDS-PAGE Western blot analysis showing visualization of coupling ofantibody 13D12 with isolated native HA;

FIG. 9B is a xerographic reproduction of a digital image showingSDS-PAGE Western blot analysis showing visualization of coupling ofantibody 41D3 with isolated native HA;

FIG. 10 is a set of histograms generated from flow cytometric analysisshowing binding and internalization of a particular sialoadhesin bindingmoiety;

FIG. 11 is a xerographic reproduction of a digital image showingSDS-PAGE and Coomassie blue staining of different samples taken duringthe antibody-saporin conjugation protocol; and

FIG. 12 is a graph showing mean immuno-peroxidase monolayer assay titersof pigs immunized with 13D12-HA or 41D3-HA.

FIG. 13. Increase of sialoadhesin expressing cells early in disease andthroughout rheumatoid arthritis disease progression in synovial tissue.Semi-quantitative scores on a four-point-scale of Sn expression in thelining layer (a) and sublining layer (b) of synovial tissue biopts.Groups include control persons, patients with UA (undifferentiatedarthritis), ERA (early RA) and Mtx res RA (Methotrexate resistant RA).Black lines represent the group medians. Statistical significantdifferences were calculated by Kruskal Wallis tests, succeeded bymultiple Mann Whitney U tests and corrected for multiple testing by HOLMprocedure with * indicating p<0.05; ** p<0.01 and *** p<0.001.

FIG. 14. Monoclonal anti-Sn antibody conjugated with Methotrexatekinetics in mouse blood. Detection by ELISA of the anti-Sn antibody atseveral time-points after intraperitoneal injection of 200 μg Ab-MTX,indicating 11% of the initial amount of conjugate is still present inthe blood after 1 week.

FIG. 15. Monoclonal anti-Sn antibody conjugated with a low dose ofMethotrexate prevents symptoms of arthritis during collagen inducedarthritis in mice. The average clinical score (a) and the incidence ofarthritis (b) of all mice in the four groups are represented at theindicated days after induction of arthritis with collagen. Groupsinclude MTX high dose (methotrexate 35 mg/kg); ab-MTX (200 g anti-Snantibody conjugated with methotrexate with an equivalent dose of 0.2mg/kg methotrexate); iso-MTX (same but irrelevant isotype as anti-Snantibody conjugated to the same amount of methotrexate); PBS.Longitudinal clinical scores, determined by mixed model analysis, weresignificantly (p<0.0001) different between PBS and MTX; PBS and ab-MTX;iso-MTX and ab-MTX; iso-MTX and MTX indicating a significant effect ofthe treatment over time.

DETAILED DESCRIPTION

Few effective targeted delivery compositions and methods are capable ofdelivering a desired cargo to a targeted cell. A particular receptor,sialoadhesin (Sn), is identified as a target for targeted deliverycompositions and methods herein.

Sialoadhesin, also called sheep erythrocyte receptor (SER) or sialicacid binding immunoglobulin-like lectin 1 (Siglec-1) belongs to theSiglec family of 1-type lectins and is expressed exclusively on subsetsof macrophages that are found mostly in spleen, lymph nodes, bonemarrow, liver, colon and lungs but not on blood monocytes as describedin P. R. Crocker et al., 1991, Embo. J. 10:1661-9; P. R. Crocker et al.,1994, Embo. J. 13:4490-503; X. Duan et al., 1998, J. Virol. 72:4520-3;A. Hartnell et al., 2001, Blood 97:288-96; and N. Vanderheijden et al.,2003, J. Virol. 77:8207-15. High Sn expression has also been detected oninflammatory macrophages in tissues from patients with rheumatoidarthritis, and on infiltrating macrophages that make close contact withbreast carcinoma cells as described in A. Hartnell et al., 2001, Blood97:288-96; and D. Nath et al., 1999, Immunology 98:213-9. Sialoadhesin(Sn) was initially identified as a sialic acid dependent-sheeperythrocyte receptor (SER) on resident bone marrow cells of mice, and isnow also characterized in a number of mammals including human, rat andswine, described in P. R. Crocker and S. Gordon, 1989, J. Exp. Med.169:1333-46; P. R. Crocker and S. Gordon, 1986, J. Exp. Med.164:1862-75; and N. Vanderheijden et al., 2003, J. Virol. 77:8207-15.

A conjugate composition is provided herein which includes a sialoadhesinbinding moiety conjugated to a cargo moiety. Conjugate compositionsincluding a sialoadhesin binding moiety conjugated to a cargo moiety maybe used to deliver a cargo moiety to a sialoadhesin expressing cell.

The term “nucleic acid” as used herein refers to RNA or DNA moleculeshaving more than one nucleotide in any form including single-stranded,double-stranded, oligonucleotide or polynucleotide. The term “nucleotidesequence” is used to refer to the ordering of nucleotides in anoligonucleotide or polynucleotide in a single-stranded form of nucleicacid. It is appreciated that, due to the degeneracy of the genetic code,multiple nucleic acids encode an identical polypeptide.

The terms “protein,” “polypeptide” and “peptide” are usedinterchangeably herein to refer to two or more amino acids linked bypeptide bonds. The term protein includes modified proteins and peptidesexemplified by, but not limited to, glycosylated, phosphorylated,ubiquitinated, myristoylated, palmitoylated, and acetylated proteins andpeptides.

The term “expression construct” refers to a recombinant or syntheticnucleic acid including a nucleic acid encoding a protein, and one ormore regulatory polynucleotides operably linked to the nucleic acidencoding the protein that direct transcription of at least the nucleicacid encoding the protein in a cell.

The term “transfection” refers to introduction of an exogenous nucleicacid into a cell.

The term “operably linked” refers to a nucleic acid in functionalrelationship with a second nucleic acid. In general, operably linkednucleic acids are contiguous. An exception is operable linkage of anenhancer, which may be non-contiguous and in functional relationshipwith another nucleic acid.

The term “vaccine” refers to a pharmaceutical composition including atleast one antigen that stimulates an immune response in a subject.

The term “vaccination” refers to administration of a vaccine tostimulate an immune response in a subject. Vaccination of a subject maybe performed to prevent or treat a disease in the subject.

The term “antigen” refers to a molecule that includes one or moreepitopes that stimulate an antigen-specific response by a component of ahost immune system, such as an immune cell. An antigen can includepeptide, proteins, glycoproteins, polysaccharides, lipids, gangliosides,portions thereof, and combinations thereof.

The term “stimulation of an immune response” means eliciting orenhancing an immune response.

The term “homologue” refers to a protein characterized by an amino acidsequence and/or structural homology to a reference protein. In general,a homologue of the reference protein is at least 50%, preferably atleast 75%, more preferably at least 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or greater, identical to the reference protein.A homologue is illustratively an orthologue of the reference proteinisolated from another species. A homologue includes a protein having oneor more amino acid substitutions, deletions or insertions compared withthe reference protein.

The term “biologically active homologue” of a reference protein refersto a protein characterized by an amino acid sequence and/or structuralhomology to the reference protein which has substantially similarfunctional, structural, and/or biochemical properties compared to thereference protein, particularly the naturally occurring referenceprotein.

One type of homologue is a conservatively modified protein and/orfragment thereof. A conservatively modified protein or fragment thereofis a protein or peptide which includes substitution of an amino acidwith a chemically similar amino acid. For example, each amino acid maybe described as having one or more of the following characteristics:electropositive, electronegative, aliphatic, aromatic, polar,hydrophobic and hydrophilic. A conservative substitution is asubstitution of one amino acid having a specified structural orfunctional characteristic for another amino acid having the samecharacteristic. Acidic amino acids include aspartate and glutamate;basic amino acids include histidine, lysine and arginine; aliphaticamino acids include isoleucine, glycine, leucine and valine; aromaticamino acids include phenylalanine, tyrosine and tryptophan; polar aminoacids include aspartate, glutamate, histidine, lysine, asparagine,glutamine, arginine, serine, threonine and tyrosine; hydrophobic aminoacids include alanine, cysteine, phenylalanine, glycine, isoleucine,leucine, methionine, proline, valine, tyrosine and tryptophan; andhydrophilic amino acids include asparagine, aspartate, glutamine,glutamate, histidine, serine and threonine. Amino acids may also bedescribed in terms of relative size, alanine, cysteine, aspartate,glycine, asparagine, proline, threonine, serine, valine, all typicallyconsidered to be small.

Percent identity is determined by comparison of amino acid orpolynucleotides, including a reference sequence and a putative homologuesequence. Algorithms used for determination of percent identityillustratively include the algorithms of S. Karlin and S. Altshul, PNAS,90:5873-5877, 1993; T. Smith and M. Waterman, Adv. Appl. Math.2:482-489, 1981, S. Needleman and C. Wunsch, J. Mol. Biol., 48:443-453,1970, W. Pearson and D. Lipman, PNAS, 85:2444-2448, 1988 and othersincorporated into computerized implementations such as, but not limitedto, GAP, BESTFIT, FASTA, TFASTA; and BLAST, publicly available from theNational Center for Biotechnology Information, for instance, on theWorld Wide Web at ncbi.nlm.nih.gov.

Sialoadhesin Binding Moiety

A sialoadhesin binding moiety binds specifically to sialoadhesin. Theterm “binds specifically” as used herein is intended to indicate that asialoadhesin binding moiety included in a conjugate interactspreferentially with sialoadhesin and does not significantly interactwith other proteins or other molecules. A sialoadhesin binding moietyconjugated to a cargo moiety has sialoadhesin-specific binding activityand thus confers sialoadhesin-specific binding activity on a conjugate.In particular, a sialoadhesin binding moiety conjugated to a cargomoiety binds to an extracellular portion of sialoadhesin expressed by acell. Further, a sialoadhesin binding moiety binds specifically withsialoadhesin present in the cell membrane of a target cell andstimulates uptake of a conjugate into the cell

In certain embodiments, a sialoadhesin binding moiety is an antibody.The term “antibody” refers to polyclonal antibodies, monoclonalantibodies (mAbs), chimeric antibodies, humanized antibodies, as well asantigen binding antibody fragments and molecules having antigen bindingfunctionality.

The term “antibody” includes an intact immunoglobulin having fourpolypeptide chains, two heavy (H) chains and two light (L) chains linkedby disulfide bonds. The term “antibody” also includes sialoadhesinbinding antibody fragments illustratively including, but not limited to,such fragments as an Fab fragment, an Fab′ fragment, an F(ab′)2fragment, an Fd fragment, an Fv fragment, an scFv fragment, and a domainantibody (dAb).

An anti-sialoadhesin antibody and/or sialoadhesin binding antibodyfragment included in a conjugate hereof is capable of bindingsialoadhesin and stimulating uptake of the conjugate into the cell.

An antibody or antibody fragment included in a conjugate hereofspecifically binds to sialoadhesin. A preferred sialoadhesin bindingmoiety binds sialoadhesin with greater affinity than it binds anothermember of the Siglec family.

A preferred sialoadhesin binding moiety included in a conjugate ischaracterized by specific binding activity for sialoadhesin of at leastabout 1×10⁵ M⁻¹. In further embodiments, a preferred sialoadhesinbinding moiety has a specific binding affinity for sialoadhesin of atleast about 1×10⁶ M⁻¹. In still further embodiments, a preferredsialoadhesin binding moiety has a specific binding affinity forsialoadhesin of at least about 1×10⁷ M⁻¹.

Anti-sialoadhesin antibodies and sialoadhesin binding antibody fragmentsmay be provided by any method, illustratively including, but not limitedto, immunization, isolation and purification, enzymatic cleavage of anintact immunoglobulin, chemical synthesis of a desired sialoadhesinbinding peptide or protein, production by recombinant nucleic acidtechnology. Combinations of such methods may also be used.

An anti-sialoadhesin antibody can be made by immunization using as anantigen a full length sialoadhesin or a peptide fragment ofsialoadhesin. Such proteins and peptides may be, illustratively a human,pig, sheep, rat, mouse, or other sialoadhesin protein or peptide.Exemplary human, mouse and porcine sialoadhesin protein sequences andpolynucleotides encoding human, mouse and porcine sialoadhesins includedherein as SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9, and SEQ ID NO:10.

Extracellular portions of sialoadhesin from various species have beencharacterized, as have sialic acid binding sites, as exemplified in D.Nath et al., J. Biol. Chem., 270:26184-26191, 1995; M. Vinson et al., J.Biol. Chem., 271:9267-9272, 1996; A. Hartnell et al., Blood, 97:288-296;and N. Vanderheijden et al., 2003, J. Virol. 77:8207-15. For example, anextracellular portion of human sialoadhesin extends from amino acid1-1642, an extracellular portion of porcine sialoadhesin extends fromamino acid 1-1643 and an extracellular portion of mouse sialoadhesinextends from amino acid 1-1638, each with reference to the sequencesdescribed herein. A sialoadhesin fragment used as an antigen inpreparation of a sialoadhesin binding antibody preferably includes oneor more Ig-like domains.

Antigens may be prepared by any of various methods, including isolationfrom natural sources, recombinant production or by chemical synthetictechniques. Sialoadhesin proteins and peptides for use as antigens inpreparation of a sialoadhesin binding antibody are similarly prepared byany of various techniques.

A peptide portion of a sialoadhesin or other antigen may be made moreimmunogenic if desired by linkage to a carrier molecule such bovineserum albumin or keyhole limpet hemocyanin. Such a linkage may beaccomplished by any of various techniques, illustratively including, butnot limited to, conjugation and expression of a fusion protein.

Recombinantly expressed proteins and peptides, such as, but not limitedto, sialoadhesin and sialoadhesin fragments, may be tagged to allow foreasier isolation. For instance, such proteins and peptides may beFc-tagged.

Antibodies, antigen binding fragments and methods for their generationare known in the art and such antibodies, antigen binding fragments andmethods are described in further detail, for instance, in AntibodyEngineering, R. Kontermann and S. Dübel (Eds.), Springer, 2001; E.Harlow and D. Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, 1988; F. Ausubel et al. (Eds.), Short Protocols inMolecular Biology, Wiley, 2002, particularly chapter 11; J. D. Pound(Ed.) Immunochemical Protocols. Methods in Molecular Biology, HumanaPress; 2nd ed., 1998; B. K. C. Lo (Ed.), Antibody Engineering Methodsand Protocols. Methods in Molecular Biology, Humana Press, 2003; and G.Kohler and C. Milstein, Nature, 256:495-497 (1975).

The term “antigen” in the context of making a sialoadhesin bindingmoiety refers to sialoadhesin or an antigenic peptide portion thereof.In a particular embodiment, an antigenic portion of sialoadhesinincludes a portion of sialoadhesin present external to a cell expressingsialoadhesin. Such a portion preferably includes a sialic acid bindingdomain.

An antibody which is a sialoadhesin binding moiety may be made using anative sialoadhesin, such as exemplified by amino acid sequencesappended hereto, and/or peptide fragments thereof, as an antigen. Anantibody which is a sialoadhesin binding moiety may be also be madeusing a sialoadhesin homologue, modified sialoadhesin and/or fragmentthereof as an antigen.

In a specific embodiment, a sialoadhesin binding moiety is a monoclonalantibody 41D3. Monoclonal antibody 41D3 (mAb 41D3) is a mouse monoclonalanti-porcine sialoadhesin antibody. Monoclonal antibody 41D3 isdescribed in N. Vanderheijden et al., 2003, J. Virol. 77:8207-15; and inX. Duan et al., 1998, J. Virol. 72:4520-3. A hybridoma producingmonoclonal antibody 41D3 was deposited with the CNCM (CollectionNationale de Cultures de Microorganisms) at the Institute Pasteur, 28,Rue du Docteur Roux, F-75724 Paris Cedex 15 and given Accession numberI-2719.

In a further specific embodiment, a sialoadhesin binding moiety is mousemonoclonal antibody 7D2 (mAb 7D2) which binds human sialoadhesin. MAb7D2 was raised against an Fc fusion protein containing the N-terminalfour domains of human sialoadhesin. MAb 7D2 is further described in A.Hartnell et al., Blood, 97:288-96, 2001 and is commercially available.

Another specific example of a sialoadhesin binding moiety is mouseanti-porcine sialoadhesin monoclonal antibody MCA2316 described, e.g.,in R. Bullido, Tissue Antigens, 1997, 49(4):403-13 and commerciallyavailable.

A sialoadhesin binding moiety is a sialoadhesin ligand in a furtherembodiment of a conjugate composition herein. As noted above,sialoadhesin is a sialic acid-binding immunoglobulin-like lectin.Sialoadhesin binds sialic acid, and in particular, α2-3 sialic acidresidues and some α2-6 and α2-8 sialic acid residues. Such sialic acidresidues illustratively include Siaα2-3Galβ1-3GalNAc;Siaα2-3Galβ1-3GlcNAc; and Siaα2-3Galβ1-4GlcNAc, Siaα2-6Galβ1-3GalNAc andSiaα2-8Neu5Acα2-3Galβ1-3GalNAc. Thus, in an embodiment in which asialoadhesin binding moiety is a sialoadhesin ligand, a sialoadhesinbinding moiety preferably includes a sialylated organic structure suchas, but not limited to, a sialylated protein or peptide, lipid, and/orcarbohydrate.

In a further embodiment, a sialoadhesin binding moiety includes anatural sialylated ligand for sialoadhesin. A natural sialylated ligandfor sialoadhesin is a sialylated structure which occurs naturally andbinds sialoadhesin in vivo. Natural sialylated ligands illustrativelyinclude CD43, galactose-type C-type lectin 1, and MUC1 antigen. Anatural sialylated ligand of sialoadhesin may be isolated from a naturalsource or recombinantly produced for inclusion in a conjugatecomposition herein.

A further natural sialoadhesin ligand is a porcine arterivirus protein.

Cargo Moiety

As noted above, a conjugate composition herein includes a sialoadhesinbinding moiety and a cargo moiety. A cargo moiety is a substance to bedelivered to a target cell.

In certain embodiments, a cargo moiety is a stimulator of a response ina target cell. For instance, a cargo moiety may be a stimulator of animmune response in a macrophage. A cargo moiety may also be a stimulatorof nitric oxide production in a target cell.

Examples of cargo moieties which are macrophage stimulatorsillustratively include interleukin-4, interleukin-10, interleukin-13,macrophage stimulating protein, CD40 ligand, and interferon-gamma.Additional stimulators include lipoteichoic acid, muramyl tripeptideTNF-alpha, GM-CSF, a lipopolysaccharide and an extracellular matrixprotein.

In a particular example, a cargo moiety which is a stimulator of animmune response is an antigen. An antigen included in a conjugate may beany type of antigen, illustratively including, but not limited to, apeptide, a protein, a lipid, a carbohydrate, and combinations of theseor other antigenic substances. An antigen may be derived from any sourceand thus may be an isolated natural substance, a recombinantly producedsubstance, a chemically synthesized substance, or otherwise provided.The identity of the antigen will depend on the desired result. Ingeneral, an antigen is included as a cargo moiety to be delivered to anantigen presenting cell in order to stimulate the immune system of asubject to produce an immune response to the antigen.

In certain embodiments, an antigen included as a cargo moiety is aporcine arterivirus peptide or protein.

Also provided is a conjugate including a cargo moiety which is aninhibitor of a target cell. Exemplary inhibitors include inhibitors ofmacrophage activation, inhibitors of inflammation and general cellinhibitors.

Inhibitors of macrophage activation and inflammation are useful as cargomoieties to decrease macrophage activation and inflammation whereproblematic, such as in autoimmune diseases illustratively including,but not limited to, endotoxemia, multiple sclerosis, rheumatoidarthritis, and lupus erythematosus. Inhibitors of macrophage activationand inflammation include anti-inflammatory cytokines andanti-inflammatory compounds such as, but not limited to, IL-10,TGF-beta, 6-(methylsulfinyl)hexyl isothiocyanate, and sesquiterpenechromones including those isolated from Ferula fukanensis.

In a further embodiment, a cargo moiety which is an inhibitor of atarget cell is a cytotoxic agent. A cytotoxic agent may be included in aconjugate for delivery to a cell in order to inhibit or destroy thecell. For example, a macrophage may be targeted for inhibition ofdestruction by a cytotoxic agent in order to inhibit a macrophageactivity, such as an inflammatory activity. In a further example, acytotoxic agent is delivered to a sialoadhesin expressing cell in orderto inhibit a microbial infection. A cytotoxic agent may be any cytotoxicagent which can be conjugated with a sialoadhesin binding moiety toproduce a conjugate hereof.

Exemplary cytotoxic cargo moieties are drugs used as anti-tumoralagents. Anti-tumoral agents are described, e.g., in Goodman et al.,Goodman and Gilman's The Pharmacological Basis of Therapeutics, 8th Ed.,Macmillan Publishing Co., 1990.

Such drugs illustratively include acivicin, aclarubicin, acodazole,acronine, adozelesin, aldesleukin, alitretinoin, allopurinol,altretamine, ambomycin, ametantrone, amifostine, aminoglutethimide,amsacrine, anastrozole, anthramycin, arsenic trioxide, asparaginase,asperlin, azacitidine, azetepa, azotomycin, batimastat, benzodepa,bicalutamide, bisantrene, bisnafide dimesylate, bizelesin, bleomycin,brequinar, bropirimine, busulfan, cactinomycin, calusterone,capecitabine, caracemide, carbetimer, carboplatin, carmustine,carubicin, carzelesin, cedefingol, celecoxib, chlorambucil, cirolemycin,cisplatin, cladribine, crisnatol mesylate, cyclophosphamide, cytarabine,dacarbazine, dactinomycin, daunorubicin, decitabine, dexormaplatin,dezaguanine, dezaguanine mesylate, diaziquone, docetaxel, doxorubicin,droloxifene, droloxifene, dromostanolone, duazomycin, edatrexate,eflomithine, elsamitrucin, enloplatin, enpromate, epipropidine,epirubicin, erbulozole, esorubicin, estramustine, estramustine,etanidazole, etoposide, etoposide, etoprine, fadrozole, fazarabine,fenretinide, floxuridine, fludarabine, fluorouracil, fluorocitabine,fosquidone, fostriecin, fulvestrant, gemcitabine, gemcitabine,hydroxyurea, idarubicin, ifosfamide, ilmofosine, interleukin II (IL-2,including recombinant interleukin II or rIL2), interferon alfa-2a,interferon alfa-2b, interferon alfa-n1, interferon alfa-n3, interferonbeta-I a, interferon gamma-I b, iproplatin, irinotecan, lanreotide,letrozole, leuprolide, liarozole, lometrexol, lomustine, losoxantrone,masoprocol, maytansine, mechlorethamine hydrochloride, megestrol,melengestrol acetate, melphalan, menogaril, mercaptopurine,methotrexate, methotrexate, metoprine, meturedepa, mitindomide,mitocarcin, mitocromin, mitogillin, mitomalcin, mitomycin, mitosper,mitotane, mitoxantrone, mycophenolic acid, nelarabine, nocodazole,nogalamycin, ormnaplatin, oxisuran, paclitaxel, pegaspargase,peliomycin, pentamustine, peplomycin, perfosfamide, pipobroman,piposulfan, piroxantrone hydrochloride, plicamycin, plomestane,porfimer, porfiromycin, prednimustine, procarbazine, puromycin,puromycin, pyrazofurin, riboprine, rogletimide, safingol, safingol,semustine, simtrazene, sparfosate, sparsomycin, spirogermanium,spiromustine, spiroplatin, streptonigrin, streptozocin, sulofenur,talisomycin, tamoxifen, tecogalan, tegafur, teloxantrone, temoporfin,teniposide, teroxirone, testolactone, thiamiprine, thioguanine,thiotepa, tiazofurin, tirapazamine, topotecan, toremifene, trestolone,triciribine, trimetrexate, triptorelin, tubulozole, uracil mustard,uredepa, vapreotide, verteporfin, vinblastine, vincristine sulfate,vindesine, vindesine, vinepidine, vinglycinate, vinleurosine,vinorelbine, vinrosidine, vinzolidine, vorozole, zeniplatin, zinostatin,zoledronate, and zorubicin. A cytotoxic cargo moiety may also bepharmaceutically acceptable salts, esters, amides, hydrates, and/orprodrugs of any of these or other cytotoxins.

In a further specific example, a cytotoxic cargo moiety is the cytotoxicribosome-inactivating protein saporin.

In some embodiments, a toxic agent may be included to inhibit or destroya pathological microbial organism associated with the cell. For example,bacteria, viruses and protozoa are known to be sequestered withincertain cells. Pathogens, illustratively including, but not limited to,Trypanosoma cruzi, Mycobacterium tuberculosis, Salmonella sp., Neisseriameningitidis, HIV, and Ross River virus, can hide in macrophages fromthe host's immune system and thereby cause persistent infections asdescribed in S. Aquaro et al., 2002, Antiviral Res. 55:209-25; I. E.Brodsky et al., 2005, Mol. Microbiol. 55:954-72; C. Jones et al., 2003,Mol. Microbiol. 49:1213-25; D. M. Monack et al., 2004, J. Exp. Med.199:231-41; V. G. Monteiro et al., 2005, Parasitol. Res. 97:380-5; J.Rengarajan et al., 2005, Proc. Natl. Acad. Sci. U.S.A. 102:8327-32; andS. J. Way et al., 2002, Virology 301:281-92. A fungus is a furtherexample of a pathogen which may be present in a host immune system.Thus, in one embodiment of a conjugate composition herein, a toxic agenteffective to inhibit an organism is a cargo moiety delivered to a cellinfected by the organism. Such toxic agents illustratively include anantibacterial agent, an antiviral agent, an antifungal agent and anantiprotozoal agent.

Specific examples of antibacterial agents include tetracyclines such as,but not limited to, doxycycline, tetracycline oxytetracycline,demeclocycline, and minocycline; beta-lactams such as, but not limitedto, penicillins and cephalosporins; aminoglycosides such as, but notlimited to, gentamycin, neomycin and streptomycin; macrolides such as,but not limited to, azithromycin, clarithromycin, lincomycin anderythromycin; nitroimidazoles such as, but not limited to, metronidazoleand tinidazole; quinolones such as, but not limited to, cinoxacin,ciprofloxacin, norfloxacin, ofloxacin, and levofloxacin; rifampin,vancomycin, and clindamycin.

Specific examples of antiviral agents include abacavir, acyclovir,amprenavir, aplaviroc, atazanavir, brecanavir, darunavir, delavirdine,dexelvucitabine, didanosine, disoproxil, efavirenz, emtricitabine,enfuvirtide, etravirine, famciclovir, fosamprenavir, ganciclovir,indinavir, lamivudine, lopinavir, maraviroc, nelfinavir, nevirapine,ritonavir, saquinavir, stavudine, tenofovir fumarate, tipranavir,vicriviroc, zalcitabine, and zidovudine.

Specific examples of antiprotozoal agents include azanidazole,chloroquine, metronidazole, nimorazole, ornidazole, secnidazole,sinefungin, tenonitrozole, temidazole, tinidazole.

Examples of antifungal agents include azoles illustratively including,but not limited to, miconazole, ketonazole, itraconazole, fluconazole,voriconazole, posaconazole, ravuconazole, terconazole, clotrimazole,sertaconazole, econazole, and fenticonazole; and polyenes illustrativelyincluding, but not limited to, natamycin, filipin, nystatin andamphotericin B.

A cargo moiety is a nucleic acid in particular embodiments. A cargonucleic acid may be DNA, RNA, a polynucleotide, an oligonucleotide, anantisense polynucleotide or oligonucleotide, or siRNA for example. Thenucleic acid may encode a protein or peptide, such as an mRNA. A cargonucleic acid may be linear, circular, supercoiled, single stranded, ordouble, triple or quadruple stranded.

In particular embodiments, a cargo nucleic acid includes an expressionconstruct. Delivery of a conjugate including a sialoadhesin bindingmoiety and a cargo nucleic acid expression construct to a cellexpressing sialoadhesin allows expression of an expression constructencoded protein or peptide in the cell.

In further embodiments, a cargo moiety which is an inhibitor orstimulator of a target cell may be a nucleic acid. A nucleic acidinhibitor may encode an inhibitor or stimulator for example.Alternatively, the nucleic acid itself may act as a stimulator orinhibitor.

A nucleic acid cargo is an inhibitor in certain embodiments, deliveredto a sialoadhesin expressing cell in order to inhibit expression of aprotein, and/or transcription and/or translation of a nucleic acid.Illustrative examples of nucleic acid inhibitors include siRNA, anantisense polynucleotide, an antisense oligonucleotide, and a ribozyme.Nucleic acid inhibitors may contain naturally occurring nucleic acidsand/or may contain modified nucleic acids such as, but not limited to,phosphorothioates.

Preparation of nucleic acid inhibitors such as these are known in theart, as described, e.g., in S. T. Crooke, Antisense Drug Technology:Principles. Strategies, and Applications, CRC Press, 2001; and D.Engelke, RNA Interference (RNAi): The Nuts & Bolts of siRNA Technology,DNA Press, 2004.

A nucleic acid inhibitor is delivered to inhibit a desired target in asialoadhesin expressing cell in vitro, ex vivo and/or in vivo,particularly a macrophage. For example, a nucleic acid inhibitor offunction or synthesis of a microbial protein or nucleic acid infectingthe sialoadhesin expressing cell is delivered to inhibit microbialinfection.

In a further example, a nucleic acid inhibitor is delivered to inhibit aprocess or function of the sialoadhesin expressing cell. For example, itmay be desired to inhibit or eliminate a cell expressing sialoadhesin.Inflammation and/or macrophage activation are processes or functions ofa sialoadhesin expressing cell that may be inhibited. An inhibitorynucleic acid cargo, such as a nucleotide analog, may be delivered toinhibit or eliminate such a cell.

A nucleic acid cargo is a stimulator in certain embodiments, deliveredto a sialoadhesin expressing cell in vitro, ex vivo and/or in vivo inorder to stimulate a process or function of the sialoadhesin expressingcell. For example, a nucleic acid cargo includes a plasmid encoding apeptide or protein to which an immune response is desired. The plasmidcargo is delivered to a sialoadhesin expressing macrophage in anorganism wherein the peptide or protein is produced and stimulates animmune response.

A plasmid encoding a peptide or protein is preferably an expressionconstruct containing a nucleic acid encoding the peptide or proteinalong with one or more regulatory polynucleotides required or desirablefor expression of the peptide or protein. Such regulatory sequencesillustratively include a promoter, an enhancer, a splicing signal, atranscription start site, a transcription termination signal, apolyadenylation signal, an internal ribosome entry site (IRES) andcombinations thereof. Suitable promoters include constitutively activepromoters, inducible promoters and cell-type specific promoters.

A nucleic acid cargo may be conjugated to a sialoadhesin binding moietydirectly or indirectly.

For example, a nucleic acid may be conjugated to a sialoadhesin bindingmoiety forming a bond between the nucleic acid and the sialoadhesinbinding moiety. For example, a carbodiimide, such as1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), maybe used to form a phosphate ester with a 5′ terminal phosphate grouppresent on a nucleic acid and then coupled with an amine group of asialoadhesin binding moiety to produce a conjugate including aphosphoramidate linkage.

In a further embodiment, a nucleic acid is indirectly conjugated to asialoadhesin binding moiety, e.g., through a linker or other molecule. Asialoadhesin binding moiety may, e.g., be conjugated to a positivelycharged protein. The positively charged protein may be brought intocontact with a nucleic acid to allow charge-based bonding between thepositively charged protein and the negatively charged nucleic acid.Examples of positively charged proteins in this context includeprotamine and polylysine.

A cargo moiety may include a microorganism and/or an antigenic moleculederived from such an organism. A cargo moiety may be, e.g. a virus, abacterium, a protozoan, and/or an antigenic molecule derived from suchan organism. A microorganism included in such a conjugate may beinactivated.

In certain embodiments, a viral cargo moiety is an intact virus orportion thereof conjugated to a sialoadhesin binding antibody. Such avirus may be any type of virus, including viruses useful in stimulatingan antigenic response to the virus.

In particular embodiments, a virus included in a conjugate as a cargomoiety is a swine viral disease virus. Swine viral disease virusesinclude PRRSV, Porcine circovirus type 2, Parvovirus and Pseudorabiesvirus. In particular embodiments, a swine viral disease virus isincluded as a cargo moiety in a conjugate herein for administration tostimulate an immune response to the virus. A particular swine viraldisease virus protein or antigenic portion of a swine viral diseasevirus protein is included in a conjugate herein as a cargo moiety inparticular embodiments. For example, a PRRSV membrane protein GP3, GP4,GP5 or Matrix (M) and/or an antigenic portion thereof is a cargo moietyin some embodiments of a conjugate. In further embodiments, a cargomoiety is a Porcine circovirus type 2 Capsid protein (CAP), a ParvovirusCapsid protein VP2 and/or a Pseudorabies virus gB, gC and/or gD protein,and/or an antigenic portion thereof. A combination of viral proteinsand/or antigenic portions thereof may be included as a cargo moiety inembodiments of a conjugate hereof.

PRRSVs are exemplified by European type PRRSV Lelystad virus, AccessionNo. M96262 and American type PRRSV VR-2332, Accession No. U87392.

In further embodiments, a virus included in a conjugate as a cargomoiety is a human viral disease virus. Human viral disease virusesHerpes simplex virus type 1, Herpes simplex virus type 2, VaricellaZoster Virus, Cytomegalovirus, Measles virus, Mumps virus, Rubellavirus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Humanimmunodeficiency virus (HIV), Poliovirus, Human papillomavirus, andCoronaviruses. In particular embodiments, a human viral disease virus isincluded as a cargo moiety in a conjugate herein for administration tostimulate an immune response to the virus. A particular human viraldisease virus protein or antigenic portion of a human viral diseasevirus protein is included in a conjugate herein as a cargo moiety inparticular embodiments. For example, a cargo moiety may be a Herpessimplex virus type 1 gB, gC and/or gD protein; a Herpes simplex virustype 2 gB, gC and/or gD protein; a Varicella Zoster Virus gH:gL complex,gB, and/or gC protein; a Cytomegalovirus gM:gN complex and/or gBprotein; a Measles virus Hemagglutinin protein (H) and/or fusion protein(F); a Mumps virus Hemagglutinin-Neuraminidase protein (HN) and/orfusion protein (F); a Rubella virus Envelope protein E1 and/or E2; aHepatitis A virus Capsid protein VP1 and/or VP2; a Hepatitis B virusEnvelope protein S, M, L and/or HbsAb; a Hepatitis C virus Envelopeglycoproteins E1 and/or E2; a Human immunodeficiency virus (HIV) gp120;a Poliovirus VP1, VP2 and/or VP3 protein; a Human papillomavirus L1protein; a Coronavirus spike protein, such as, but not limited to, SARSCoronavirus Spike protein (S); and/or an antigenic portion of any ofthese. In particular embodiments, Human papillomavirus L1 protein is aHuman papillomavirus type 16, 18, 6 and/or 11 L1 protein and/or anantigenic portion thereof. A combination of viral proteins may beincluded as a cargo moiety in embodiments of a conjugate hereof.

Influenza viruses are a major cause of human and animal disease.Influenza viruses are classed and named according to the specificcharacteristics of two proteins on the surface of the virus,hemagglutinin (also called hemagglutinin) and neuraminidase. At leastsixteen different influenza virus subtypes have been identifiedaccording to hemagglutinin protein characteristics. These subtypes arecalled H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15and H16. Numerous influenza virus strains of each subtype have beenidentified and many have been characterized by nucleic acid sequencingand/or protein sequencing of the viral glycoprotein hemagglutinin.Nucleotide and protein sequences of the influenza virus proteinhemagglutinin are known in the art and are available to the public viathe National Center for Biotechnology Information (NCBI) Entrez proteinand nucleotide search and retrieval system which have been compiled froma variety of sources, including GenBank, RefSeq, and PDB, and includingSwissProt, PIR, PRF, PDB, genpept and translations from annotated codingregions in GenBank and RefSeq under accession numbers included herein.The protein and polynucleotides associated with the accession numbersincluded herein characterize influenza virus hemagglutinins suitable forinclusion as a cargo moiety in conjugates herein.

In particular, peptides of influenza virus subtype H1 hemagglutininsuitable for inclusion as a cargo moiety in conjugates herein have thefollowing accession numbers and are available to the public via theNational Center for Biotechnology Information (NCBI) Entrez proteinsearch and retrieval system:

AAF87274; AAF87282; AAF87284; AAN64900; AAN64902; AAN83988; AAQ10372;AAQ10387; AAQ10394; AAT81327; AAT81329; AAT81330; AAT81336; AAT81338;ABB9551; ABB19618; ABB79979; ABC02277; ABD62781; ABD94965; ABD95075;ABD95152; ABD95229; ABD95284; ABD95306; ABF47869; ABF82896; ABG66974;ABI21200; ABI21222; ABJ09327; ABK40028; ABO21716; ABO21724; ABO32992;ABP49327; ABP49382; ABP49481; BAE53729; BAE96533; BAE96534; BAE96536;BAF03629; CAA40728; AAF87278; AAF87279; AAF87280; AAF87281; AAN64903;AAN64904; AAN64905; AAQ10390; AAQ10391; AAR90881; AAT81331; AAT81332;AAT81333; AAT81334; AAT81335; AAT93388; AAY56898; ABA43189; ABB19562;ABB21772; ABC86237; ABD61518; ABD62843; ABD79255; ABD94987; ABD95240;ABD95251; ABD95273; ABF47891; ABF82662; ABF82684; ABF82841; ABF82863;ABF82918; ABI84478; ABI84948; ABI93028; ABJ16686; ABK40006; ABK40557;ABN50917; ABN51066; ABN51088; ABO21709; ABO21723; CAA40730; CAA86563;CAA91082; AAA16808; AAA16813; AAA16880; AAA19935; AAA43142; AAA43153;AAA43168; AAA43170; AAA43171; AAA43175; AAA43231; AAA43240; AAC57415;AAK40318; AAK51344; AAK51345; AAK51347; AAN46827; AAN64894; AAN64896;AAP69688; AAP69691; AAP79971; AAU09400; ABA08519; ABA42247; ABB03123;ABB04972; ABB80045; ABB84190; ABB86887; ABD77675; ABD78071; ABD94789;ABE12248; ABF47693; ABG72868; ABG72869; ABG79952; ABI54437; ABI54444;ABI54445; ABO21730; ABO21731; ABO33006; ABO52038; ABO52258; ABP49305;ABP49360; BAF31892; CAA86560; CAA86561; CAA86562; CAC18524; AAA43158;AAA43169; AAA43173; AAA43176; AAA43190; AAA43194; AAA43225; AAA43232;AAA43283; AAK40314; AAK40315; AAK51348; AAN64897; AAN64898; AAN64899;AAP79977; AAX56530; AAZ79549; ABA08497; ABA12707; ABA42258; ABB51962;ABB53603; ABB82194; ABB82205; ABB82216; ABB86877; ABB86917; ABB86929;ABB86937; ABB86946; ABC40522; ABD77708; AAA43661; AAA43680; AAF06945;AAF06946; AAF87275; AAF87276; AAF87283; AAN64901; AAQ10369; AAQ10373;AAQ10380; AAQ10385; AAQ10386; AAQ10388; AAQ10395; AAQ10396; AAT81328;AAT81337; AAT81339; AAT81340; AAT85679; ABA12729; ABB19571; ABB19607;ABB19628; ABB20429; ABB79990; ABC40631; ABD61540; ABD61735; ABD62061;ABD79101; ABD85261; ABD95053; ABD95064; ABD95086; ABD95163; ABD95218;ABD95295; ABF71860; ABF82852; ABF82874; ABF82885; ABF82907; ABI92379;ABI96088; ABI96091; ABI96093; ABI96097; ABI96098; ABI96101; ABI96107;ABI96108; ABI96111; ABJ09151; ABJ53493; ABK40510; ABK40579; ABK40601;ABM22246; ABO38384; ABO38406; BAA96109; BAA96114; BAA96115; BAA96122;BAA96124; BAA96125; CAA35094; AAA58799; AAA58801; AAA65544; AAA65546;AAA65551; AAA65553; AAA65554; AAA74285; AAA74289; AAA74291; AAA74293;AAA74299; AAA91616; AAA92279; AAB03291; AAB27052; AAB29091; AAB39351;AAB50958; AAB50966; AAG22555; ABD77917; ABD77928; ABD77950; ABD78038;ABD94778; ABE12634; ABE26991; ABF47583; ABG48049; ABG72867; ABI20826;ABI51313; ABI54438; ABI84617; ABI92302; ABI95250; ABI96094; ABI96095;ABI96096; ABI96104; ABI96105; ABI96106; ABJ53427; ABJ53504; ABK40689;ABM22224; ABO38318; ABO38340; ABO44046; BAA96111; BAA96112; BAA96117;BAA96118; BAA96121; BAA96126; BAA96127; BAA96128; BAA96131; BAF47397;AAA65547; AAA65548; AAA65549; AAA65550; AAA65556; AAA65557; ABG37362;ABG66973; ABG66975; ABI21211; ABI21233; ABJ16609; ABM21960; ABM66864;ABN50928; ABN51077; ABN59423; ABN59434; ABO21725; ABO52104; ABP49316;ABP49338; ABP49349; BAE53730; BAE96535; BAE96537; BAE96541; BAE96542;BAF03627; CAA40729; CAA82950; CAA91083; AAA 16779; AAA 16809; AAA 16812;AAA 16879; AAA 16905; AAA43161; AAA43167; AAA43172; AAA43206; AAA43209;AAA43233; AAA43234; AAA43238; AAC53845; AAC53846; AAC57166; AAK40317;AAK51342; AAK51343; AAK51346; AAF06947; AAF75994; AAF80098; AAF80099;AAF87277; AAQ10367; AAQ10368; AAQ10389; AAQ10392; AAQ10393; AAZ38627;ABA42575; ABB19574; ABB19667; ABD63063; ABD79112; ABD85123; ABD94976;ABD94998; ABD95130; ABD95141; ABD95262; ABF47880; ABF82673; ABF82819;ABF82830; ABF82929; ABG66976; ABG66977; ABG88344; ABK39995; ABK40534;ABK40546; ABK40568; ABN50756; ABN50900; ABO33025; ABO52280; ABP49393;ABP49448; BAE96538; BAE96539; BAE96540; CAA40731; CAA42444; AAL60444;AAL87869; AAL87871; AAM76686; AAM76689; AAM76690; AAP34322; AAP69678;AAP69679; AAP69681; AAZ74374; ABA08464; ABA18037; ABB19518; ABB19529;ABB19540; ABC66233; ABC66236; ABD77719; ABD77807; ABD77818; ABD77972;ABD94811; ABD95328; ABD95339; ABE11657; ABE11723; ABE11812; ABE11922;ABE12032; ABF21277; ABG26243; ABG26791; ABG26813; ABG26824; ABG26945;ABG67477; ABI21530; ABI21552; ABI21574; ABI22109; ABI30367; ABI96117;ABI96118; ABI96123; AAA67338; AAA72339; AAA74296; AAA74297; AAA74298;AAA79714; AAA79727; AAB03292; AAB39352; AAB50960; AAB50961; AAB50962;AAB50963; AAB50964; AAB50965; AAL60449; AAL87868; AAM22277; AAM22278;AAM76691; AAP34323; AAP34324; AAT65329; AAZ83977; ABA06510; ABA42324;ABC66239; ABD78093; ABD95350; ABD95712; ABE11668; ABE11690; ABE11834;ABE11856; ABE11878; ABE11889; ABF21278; ABF47561; ABF82940; ABG26835;ABG88300; ABG88311; ABG88333; ABG88542; ABI85225; AAK51352; AAN64893;AAN64895; AAP69687; AAP69692; AAP79964; AAZ79392; AAZ79538; ABA12715;ABA42280; ABB03134; ABB03145; ABB86899; ABB86907; ABC40533; ABD77939;ABD94800; ABF47605; ABF47704; ABG72870; ABG88256; ABI20848; ABI54442;ABI54443; ABI54446; ABI95217; ABI96089; ABI96090; ABI96092; ABI96099;ABI96100; ABI96102; ABI96109; ABI96110; ABO38065; ABO38362; ABO38373;ABO38395; BAA96110; BAA96113; BAA96116; BAA96123; CAA24272; CAA35097;AAA58800; ABI96114; ABI96115; ABI96120; ABI96121; ABI96122; ABI96127;ABI96130; ABI96137; ABI96140; ABI96141; ABI96145; ABI96146; ABI96147;ABI96150; ABK79959; ABL67253; ABM22202; ABO37988; ABO38010; ABO38021;AAB52910; AAB81460; AAB81463; AAD25308; AAK67319; AAK67320; AAK67325;AAK67326; AAK67327; AAK67328; AAK67335; AAK67336; AAK67337; AAK67344;AAK70451; AAK70452; AAK70453; AAK70458; AAK70459; AAK71687; AAK73325;AAK73326; AAK73331; AAK73332; AAK73333; AAA65545; AAA65552; AAA65555;AAA74286; AAA74290; AAA74292; AAA74300; AAA99877; AAB39851; AAB50957;AAB50959; AAL60443; AAL87870; AAM76687; AAM76688; AAP69676; AAP69677;AAP69680; AAP69682; ABA18145; ABB19507; ABB53729; ABB53740; ABC66234;ABC66235; ABD77730; ABD77796; ABD77961; ABD77983; ABD78082; ABD78104;ABD95317; ABE11701; ABE11712; ABE11734; ABE11823; ABE11900; ABE11942;ABF21274; ABF21276; ABF47572; ABG26242; ABG26244; ABG26245; ABG26780;AAK73334; AAK73341; AAK73342; AAK73343; AAK73344; AAL29701; AAL29707;AAO65612; AAV68006; AAW50829; AAW50830; AAW50831; AAW50832; AAY42117;AAY42118; AAY42121; AAY42122; AAZ17358; AAZ17359; AAZ79604; ABA87057;ABB02792; ABB02814; ABB02913; ABB02936; ABB83026; ABB83127; ABC66240;ABD59849; ABD60944; ABD60955; ABD78016; ABD94756; ABD95042; ABD95119;ABD95174; ABF47638; ABF47660; ABF47715; ABF47759; ABF47770; ABF47792;ABF47814; ABG37395; ABG47807; ABI96124; ABI96126; ABI96134; ABI96143;ABI96144; ABI96151; ABI96153; ABK79948; ABK80036; ABK80047; ABM22169;ABO38032; ABO52225; BAA00308; BAA00718; BAA00720; BAA01280; BAA21641;AAB52905; AAB52907; AAB57740; AAB81456; AAB81457; AAB81459; AAC14275;AAD25304; AAD25305; AAD25307; AAK67322; AAK67324; AAK67329; AAK67332;AAK67338; AAK67339; AAK67341; AAK67343; AAK70449; AAK70450; AAK70456;AAK73322; AAK73324; AAK73328; AAK73330; AAK73338; AAK73340; ABI21541;ABI21563; ABI30378; ABI85231; ABI96116; ABI96119; ABI96125; ABI96132;ABI96135; ABI96142; ABI96152; ABJ09184; ABK80025; ABO38043; BAA00309;BAA00719; BAA02765; BAA21642; AAB52904; AAB52906; AAB52908; AAB81458;AAD05215; AAD17229; AAD25303; AAD25306; AAD25312; AAK67321; AAK67323;AAK67330; AAK67331; AAK67333; AAK67340; AAK67342; AAK70455; AAK70457;AAK70464; AAK73321; AAK73323; AAK73327; AAK73329; AAK73336; AAK73337;AAK73339; AAL29694; AAK73345; AAL02002; AAL29695; AAL29702; AAL29708;AAL29710; AAL29711; AAL47668; AAO65768; AAW50828; AAW50836; AAY42114;AAY42115; AAZ15839; AAZ15840; AAZ15842; AAZ83253; ABA87080; ABA87231;ABB03101; ABB53707; ABB83015; ABC66243; ABC66246; ABD15515; ABD60779;ABD60856; ABD60878; ABD60900; ABD60933; ABD60966; ABD94943; ABD95020;ABD95097; ABD95207; ABF47748; ABF47825; ABF47847; ABG80183; ABG88212;ABI30565; ABI55088; ABI96154; ABI96160; ABI96166; ABG47829; ABI20870;ABI54447; ABI95272; ABI96155; ABI96156; ABI96157; ABI96162; ABI96163;ABI96171; ABI96172; ABI96173; ABJ51891; ABM22279; ABM66886; ABM66908;ABN51143; ABO32948; ABO32970; ABO32981; BAC82844; BAC82847; BAC82848;BAC82853; BAC82854; BAC82860; BAC82869; BAC82870; BAC82880; BAC82889;BAC82890; BAC82898; BAD02346; CAC86333; CAC86334; CAC86621; CAD29905;CAD29906; CAD29915; CAD29916; CAD29921; CAD29922; CAD29923; CAD29931;CAD29932; CAD29941; AAL29696; AAL29697; AAL29703; AAL29709; AAL29712;AAL29713; AAL47667; AAO88265; AAT00437; AAT00438; AAV67984; AAW22156;AAW50827; AAW50834; AAW50835; AAW50837; AAW56635; AAY42116; AAZ15838;AAZ15841; AAZ15843; ABA87045; ABA87091; ABB02825; ABB80103; ABB83138;ABC66244; ABC66245; ABD15258; ABD59847; ABD60867; ABD60889; ABD60911;ABD95108; ABD95185; ABD95196; ABF47726; ABF47737; ABF47836; ABG88201;ABI96159; ABI96161; ABI96165; ABI96167; ABI96168; ABI96169; ABI96170;ABJ51892; ABJ51894; ABJ51895; ABJ53449; ABK40634; ABK57093; ABL67055;ABL67066; ABL67209; ABM22026; ABM22268; ABN50940; ABN50962; ABN50973;ABO44123; BAC82843; BAC82850; BAC82851; BAC82857; BAC82859; BAC82866;BAC82867; BAC82873; BAC82876; BAC82877; BAC82879; BAC82886; BAC82887;BAC82893; BAC82896; BAC82897; BAD02356; CAC86337; CAC86605; CAC86608;CAC86609; CAC86617; CAC86618; CAC86620; CAC86625; CAD29902; CAD29909;CAD29912; CAD29918; ABJ16719; ABJ51890; ABJ51893; ABK57092; ABL67187;ABM22257; ABO44134; ABP49217; BAC82842; BAC82849; BAC82852; BAC82858;BAC82864; BAC82865; BAC82868; BAC82874; BAC82875; BAC82878; BAC82884;BAC82885; BAC82888; BAC82894; BAC82895; CAC86336; CAC86606; CAC86607;CAC86610; CAC86611; CAC86616; CAC86619; CAD29900; CAD29901; CAD29903;CAD29910; CAD29911; CAD29917; CAD29919; CAD29926; CAD29927; CAD29933;CAD29936; CAD29937; CAD29939; CAD29943; CAD29946; CAD29947; CAD29924;CAD29925; CAD29928; CAD29934; CAD29935; CAD29938; CAD29944; CAD29945;CAD29948; CAD35680; CAD35682; CAD57617; CAD57619; CAA86567; CAA91080;CAA91081; AAA16778; AAA16810; AAA16811; AAA16814; AAA16815; AAA19934;AAA43157; AAA43166; AAA43235; AAA43236; AAC57167; AAC57168; AAC57169;AAK40313; AAK40316; AAK51341; AAK51349; AAK51350; AAK51351; AAN64892;AAP69685; AAP69686; AAP69689; AAP69690; AAU25851; ABA08475; ABA08486;ABA08508; ABA12696; ABA42236; ABB96487; ABC41714; ABD78060; ABE27153;ABF47671; ABG72863; ABG72864; ABG72865; ABG72866; ABI20837; ABI20859;ABI54439; ABI54440; ABI54441; ABI84855; ABI92181; ABI92313; ABI95294;ABI96103; ABJ53438; ABJ53515; ABK40590; ABM22213; ABM22235; ABO32678;ABO38329; ABO38351; ABO52797; BAA96119; BAA96120; BAA96129; BAA96130;AAA67181; AAA67182; AAA67183; AAA74287; AAA74288; AAA74294; AAA74295;AAA92280; AAB39353; AAL87865; AAL87866; AAL87867; AAL87872; AAM75158;AAP34325; AAP60036; AAP60037; AAP69673; AAP69674; AAP69675; AAP69683;AAP69684; AAZ83299; AAZ85126; ABA06542; ABC66232; ABC66237; ABC66238;ABD77994; ABE11679; ABE11845; ABE11867; ABF21272; ABG26246; ABG37120;ABG67491; ABG88322; ABG88553; ABI21519; ABI96112; ABI96113; ABI96128;ABI96129; ABI96138; ABI96139; ABI96148; ABI96149; ABJ16675; ABK40039;ABK40050; ABK79970; ABL67264; ABM22180; ABM22191; ABN59401; ABN59412;ABO37999; ABO38054; BAA00721; BAA00722; BAA01027; BAA02766; BAA02767;BAA02768; BAA02769; BAA05874; BAA06719; AAB52909; AAB81461; AAB81462;AAD05216; CAD29942; CAD35678; CAD35679; CAD35686; CAD35687; CAD35688;CAD57616; CAD57620; CAD57623; CAD35681; CAD35683; CAD35684; CAD57618;AAD05217; AAD05218; AAD05219; AAD17218; AAD17219; AAD25301; AAD25302;AAD25309; AAD25310; AAD25311; AAK67334; AAK70454; AAK73320; AAK73335;AAL15459; AAL29693; AAL29698; AAL29699; AAL29700; AAL29704; AAL29705;AAL29706; AAL29714; AAL29715; AAO65769; AAT00436; AAT12706; AAW50833;AAY42119; AAY42120; AAY78939; AAZ15844; ABB02781; ABB02803; ABB02924;ABC42750; ABC66241; ABC66242; ABD59848; ABD78005; ABD78027; ABD95009;ABD95031; ABF47627; ABF47649; ABF47781; ABF47803; ABF47955; ABG37384;ABG47818; ABG47840; ABG80172; ABI21189; ABI95261; ABI95283; ABI96158;ABI96164; ABI96174; ABJ16642; ABJ16653; ABJ16664; ABJ16730; ABM22158;ABM22290; ABM66897; ABM67051; ABN50951; ABO32959; BAC82845; BAC82846;BAC82855; BAC82856; BAC82861; BAC82862; BAC82863; BAC82871; BAC82872;BAC82881; BAC82882; BAC82883; BAC82891; BAC82892; CAC86335; CAC86612;CAC86613; CAC86614; CAC86615; CAC86622; CAC86623; CAC86624; CAD29898;CAD29899; CAD29904; CAD29907; CAD29908; CAD29913; CAD29914; CAD29920;CAD29929; CAD29930; CAD29940; CAD29958; CAD35685; CAD57621 and CAD57622.

Protein sequences of influenza virus subtype H2 hemagglutinin suitablefor inclusion as a cargo moiety in conjugates herein have the followingaccession numbers and are available to the public via the NationalCenter for Biotechnology Information (NCBI) Entrez protein search andretrieval system:

BAC43764; BAF02312; AAO46270; AAO46271; AAO46272; AAO46273; AAO46274;AAO46275; AAO46276; AAO46277; AAO46278; AAO46279; AAO46280; AAO46281;AAO46282; AAO46283; AAO46284; AAO46285; AAO46286; AAO46287; AAO46288;AAO46289; AAO46290; AAO46291; AAO46292; AAO46293; AAO46294; AAO46295;AAO46296; AAO46297; AAO46298; AAO46299; AAO46300; AAO46301; AAO46302;AAO46303; AAO46304; AAO46305; AAS57527; AAS57528; AAS57529; AAS57530;AAT65325; AAT65327; AAT65331; AAT65348; AAT65351; AAV91219; ABB17150;ABB17670; ABB17681; ABB17692; ABB17703; ABB17714; ABB18378; ABB17725;ABB17736; ABB17756; ABB17813; ABB18025; ABB18036; ABB18047; ABB18058;ABB18069; ABB18080; ABB19639; ABB20141; ABB20229; ABB20240; ABB20466;ABB20509; ABI84382; ABI84384; ABI84450; ABI84458; ABI84459; ABI84588;ABI84744; ABI84755; ABI84959; ABI85183; ABL67022; ABM21949; ABO38098;ABO38296; ABO38307; ABO38701; ABO38712; ABO38723; ABO38734; ABO44057;ABO44090; ABO44101; ABO52236; ABO52247; ABO52302; ABO52379; ABP49437;ABP49459; ABP49470; BAA02770; BAA02771; BAA02772; BAA02773; BAA02774;BAA02775; AAY23639; AAY23640; AAY28987; AAY87410; AAY87419; ABF21270;ABF21275; AAA43185; AAA43196; AAA43089; AAA43090; AAA43659; AAA43243;AAA43117; AAA43284; AAA43450; AAA43096; AAA43247; AAA43248; AAA43088;AAA43345; AAA43576; AAA43578; AAA43658; AAA43660; AAA43662; AAA43678;AAA64362; AAA64364; AAA64365; AAA64363; AAA64366; BAF33428; BAF33438;BAF33398; BAF33408; BAF34322; BAF34377; BAF47131; BAF48641; BAF49415;AAD43235; AAD43236; AAD43237; AAD43238; AAD43239; AAD43240; AAD43241;AAD43242; AAD43243; AAD43244; AAD43245; AAD43246; AAD43247; AAD43248;AAD43249; AAK14980; AAF82100; AAF82101; AAF82102; AAF82103; AAF82104;AAF82105; AAF82106; AAF82107; AAF82108; AAF82109; AAF82110; AAF82111;AAF82112; AAN83926; AAN83927; AAN83928; AAN83929; AAN83930; AAN83931;AAO46268 and AAO46269.

Protein sequences of influenza virus subtype H3 hemagglutinin suitablefor inclusion as a cargo moiety in conjugates herein have the followingaccession numbers and are available to the public via the NationalCenter for Biotechnology Information (NCBI) Entrez protein search andretrieval system:

BAA77284; BAA77285; BAA77286; BAA77287; BAA77288; BAA77289; BAA77290;BAA77291; BAA77292; BAA77293; BAA77294; BAA86062; BAA86063; BAA86064;BAA86065; BAA96300; BAA96301; BAA96302; BAA96303; BAE75900; BAE75901;BAE75902; BAE75903; BAE75904; BAE75905; BAE75906; BAE75907; BAE75908;BAE75909; BAE75910; BAE75911; BAE75912; BAE75913; BAE75914; BAE75915;BAE75916; BAE75917; BAE75918; BAE75919; BAE54256; BAE54257; BAE54258;BAE54259; BAE54260; BAE54261; BAE54262; BAE75854; BAE94240; BAE94241;BAE94242; BAE94243; BAE94244; BAE94245; BAE94246; BAE94247; BAE94248;BAE94249; BAE94250; BAE94251; BAE94568; BAE94569; BAE94570; BAE96004;BAE96005; BAE96006; BAE96007; BAF46357; BAF46358; BAF46359; BAF46360;BAF46361; BAF46362; BAF46363; BAF46364; BAF46365; BAF46366; BAF46367;BAF46375; BAF46376; BAF46377; BAF46378; BAF46379; BAF46380; BAF46381;BAF46382; BAF46383; BAF46384; BAF46317; BAF46318; BAF46319; BAF46320;BAF46325; BAF46326; BAF46327; BAF46328; BAF46329; BAF46330; BAF46331;BAF46338; BAF46339; BAF46340; BAF46341; BAF46342; BAF46343; BAF46344;BAF47998; BAF47999; BAF48000; BAF48001; BAF48002; BAF48003; BAF48004;BAF48005; BAF48006; BAF48007; BAF48008; BAF48009; BAF48010; BAF48011;BAF48012; BAF48013; BAF48014; BAF48015; BAF48016; BAF48017; BAF48018;BAF48019; BAF48020; BAF48021; BAF48022; BAF48023; BAF48024; BAF48025;BAF48026; BAF48027; BAF48028; BAF48029; BAF48030; BAF48031; BAF48032;BAF48033; BAF48034; BAF48035; BAF48036; BAF48037; BAF48038; BAF48039;BAF33059; BAF34375; BAF34924; BAF37221; BAF43466; BAF46752; BAF46760;BAF46902; BAF46910; BAF48361; AAB66723; AAB66724; AAB66725; AAB66726;AAB66727; AAB66728; AAB66729; AAB66730; AAB66731; AAB66732; AAB66733;AAB66734; AAB66735; AAB66736; AAB66737; AAB66738; AAB66739; AAB66740;AAB66741; AAB66742; AAB66743; AAB66744; AAB66745; AAB66746; AAB66747;AAB66748; AAB66749; AAB66750; AAB66751; AAB66752; AAB66753; AAB66754;AAB66755; AAB66756; AAB66757; AAB66758; AAB66759; AAB66760; AAB66761;AAB66762; AAB66763; AAB66764; AAB66765; AAB66766; AAB66767; AAB66768;AAB66769; AAB66770; AAB66771; AAB66772; AAB66773; AAB66774; AAB66775;AAB66776; AAB66777; AAB66778; AAB66779; AAB66780; AAB66781; AAB66782;AAB66783; AAB66784; AAB66785; AAB66786; AAB66787; AAB66788; AAB66789;AAB66790; AAB66791; AAB66792; AAB66793; AAB66794; AAB66795; AAB66796;AAB66797; AAB66798; AAB66799; AAB66800; AAB66801; AAB66802; AAB66803;AAB66804; AAB66805; AAB66806; AAB66807; AAB66808; AAB66809; AAB66810;AAB63681; AAB63682; AAB63683; AAB63684; AAB63685; AAB63686; AAB63687;AAB63688; AAB63689; AAB63690; AAB63691; AAB63692; AAB63693; AAB63694;AAB63695; AAB63696; AAB63697; AAB63698; AAB63699; AAB63700; AAB63701;AAB63702; AAB63703; AAB63704; AAB63705; AAB63706; AAB63707; AAB63708;AAB63709; AAB63710; AAB63711; AAB63712; AAB63713; AAB63714; AAB63715;AAB63716; AAB63717; AAB63718; AAB63719; AAB63720; AAB63721; AAB63722;AAB63723; AAB63724; AAB63725; AAB63726; AAB63727; AAB63728; AAB63729;AAB63730; AAB63731; AAB63732; AAB63733; AAB63734; AAB63735; AAB63736;AAB63737; AAB63738; AAB63739; AAB63740; AAB63741; AAB63742; AAB63743;AAB63744; AAB63745; AAB63746; AAB63747; AAB63748; AAB63749; AAB63750;AAB63751; AAB63752; AAB63753; AAB63754; AAB63755; AAB63756; AAB63757;AAB63758; AAB63759; AAB63760; AAB63761; AAB63762; AAB63763; AAB63764;AAB63765; AAB63766; AAB63767; AAB69773; AAB69774; AAB69775; AAB69776;AAB69777; AAB69778; AAB69779; AAB69780; AAB69781; AAB69782; AAB69783;AAB69784; AAB69785; AAB69786; AAB69787; AAB69788; AAB69789; AAB69790;AAB69791; AAB69792; AAB69793; AAB69794; AAB69795; AAB69796; AAB69797;AAB69798; AAB69799; AAB69800; AAB69801; AAB69802; AAB69803; AAB69804;AAB69805; AAB69806; AAB69807; AAB69808; AAB69809; AAB69810; AAB69811;AAB69812; AAB69813; AAB69814; AAB69815; AAB69816; AAB69817; AAB69818;AAB69819; AAB69820; AAB69821; AAB69822; AAB69823; AAB69824; AAB69825;AAB69826; AAB69827; AAB69828; AAB69829; AAB69830; AAB69831; AAB69832;AAB69833; AAB69834; AAB69835; AAB69836; AAB69837; AAB69838; AAB69839;AAB69840; AAB69841; AAB69842; AAB69843; AAB69844; AAB69845; AAB69846;AAB69847; AAB69848; AAB69849; AAB69850; AAB69851; AAC59602; AAC59603;AAC59604; AAC63474; AAC63475; AAC63476; AAC63477; AAC63478; AAC31556;AAC36729; AAC36730; AAC36731; AAC36732; AAC36733; AAC36734; AAC36735;AAC36736; AAC36737; AAC36738; AAC78086; AAC78087; AAC78088; AAC78089;AAC78090; AAC78091; AAC78092; AAC78093; AAC78094; AAC78095; AAC78096;AAC78097; AAC78098; AAC83790; AAC83791; AAC83792; AAC83793; AAC83794;AAC83795; AAC83796; AAC83797; AAC83798; AAC83799; AAC83800; AAF06948;AAF06949; AAF06950; AAD34847; AAD34848; AAD34849; AAD34850; AAD34851;AAD34852; AAD34853; AAD34854; AAD34855; AAD34856; AAD34857; AAD51239;AAD51240; AAD51241; AAD51242; AAF16416; AAF16417; AAF16418; AAF16419;AAF16420; AAF16421; AAF16422; AAF16423; AAF16424; AAF16425; AAF16426;AAF16427; AAF16428; AAF16429; AAF16430; AAF16431; AAF16432; AAF16433;AAF16434; AAF16435; AAF16436; AAF16437; AAF16438; AAF16439; AAF16440;AAF16441; AAF16442; AAF16443; AAF16444; AAF16445; AAF16446; AAF16447;AAF16448; AAF16449; AAF16450; AAF16451; AAF16452; AAF16453; AAF16454;AAF16455; AAF16456; AAF16457; AAF16458; AAF16459; AAF16460; AAF16461;AAF16462; AAF16463; AAF16464; AAF16465; AAF16466; AAF16467; AAF16468;AAF16469; AAF16470; AAF16471; AAF16472; AAF16473; AAF16474; AAF16475;AAF16476; AAF16477; AAF16478; AAF16479; AAF16480; AAF16481; AAF16482;AAF16483; AAF16484; AAF16485; AAF16486; AAF16487; AAF16488; AAF16489;AAF16490; AAF16491; AAF16492; AAF16493; AAF16494; AAF16495; AAF16496;AAF16497; AAF16498; AAF16499; AAF16500; AAF16501; AAF16502; AAF16503;AAF16504; AAF16505; AAF16506; AAF16507; AAF16508; AAF16509; AAF16510;AAF16511; AAF16512; AAF16513; AAF16514; AAF16515; AAF16516; AAF16517;AAF16518; AAF22345; AAF22346; AAF22347; AAF22348; AAF22349; AAF22350;AAF22351; AAF22352; AAF22353; AAF18089; AAF18090; AAF18091; AAF18092;AAF18093; AAF13705; AAF13706; AAF19421; AAL59048; AAL59049; AAL59050;AAL59051; AAO15354; AAO15355; AAO15356; AAO15357; AAF60285; AAG01749;AAG01758; AAG01767; AAG01776; AAG01785; AAK49194; AAK49195; AAK49196;AAK49197; AAK49198; AAK49199; AAK49200; AAK49201; AAK49202; AAK49203;AAK49204; AAG10735; AAG10736; AAG10737; AAG10738; AAG10739; AAG10740;AAG33221; AAG33222; AAG33223; AAG33224; AAG47797; AAG47798; AAG47799;AAG47800; AAG47801; AAG47802; AAG47803; AAG47804; AAG47805; AAG47806;AAG47807; AAG47808; AAG47809; AAG47810; AAG47811; AAG47812; AAG47813;AAG47814; AAG47815; AAG47816; AAG47817; AAG47818; AAG47819; AAG49302;AAG49303; AAG49304; AAG49305; AAG49306; AAG49307; AAG49308; AAG49309;AAG49310; AAG49311; AAG49312; AAG49313; AAG49314; AAG49335; AAG49336;AAG49337; AAG49338; AAG49339; AAK51718; AAL18558; AAL18559; AAL18560;AAL18561; AAL18562; AAL18563; AAL18564; AAL18565; AAL18566; AAL18567;AAL18568; AAL18569; AAL18570; AAL18571; AAL18572; AAL18573; AAL18574;AAL18575; AAL18576; AAL18577; AAL18578; AAL18579; AAL18580; AAL18581;AAL18582; AAL18583; AAL18584; AAL18585; AAL18586; AAL18587; AAL18588;AAL18589; AAL18590; AAL18591; AAL18592; AAL18593; AAL18594; AAL18595;AAL18596; AAL18597; AAL18598; AAK82853; AAK82854; AAK82855; AAK82856;AAK82857; AAK82858; AAK82859; AAK82860; AAK82861; AAK82862; AAK82863;AAK82864; AAK82865; AAK82866; AAK82867; AAK82868; AAK82869; AAK52910;AAK52911; AAK52912; AAK54141; AAK54142; AAK54143; AAK54144; AAK54145;AAK54146; AAK54147; AAK54148; AAK54149; AAK54150; AAK54151; AAK63816;AAK63817; AAK63818; AAK63819; AAK63820; AAK63821; AAK63822; AAK63823;AAK63824; AAK63825; AAK63826; AAK67171; AAK67172; AAK67173; AAK67174;AAK67175; AAK67176; AAK67177; AAK67178; AAK67179; AAK67180; AAK67181;AAK67182; AAK67183; AAK67184; AAK67185; AAK67186; AAK67187; AAK67188;AAK67189; AAK67190; AAK67191; AAK67192; AAK67193; AAK67194; AAK67195;AAK67196; AAK67197; AAK67198; AAK67199; AAK67200; AAK67201; AAL30462;AAL30463; AAL30464; AAL60147; AAL60148; AAL60149; AAL60150; AAL60151;AAL60152; AAL60153; AAL77301; AAL77302; AAL77303; AAL77304; AAL77305;AAL77306; AAL77307; AAL77308; AAL77309; AAL77310; AAL77311; AAL77312;AAL77313; AAL77314; AAL77315; AAL77316; AAL77317; AAL77318; AAL77319;AAL77320; AAL77321; AAL77322; AAL77323; AAL77324; AAL77325; AAL77326;AAL77327; AAL77328; AAL77329; AAL62329; AAM46871; AAM46872; AAM46873;AAM46874; AAM46875; AAM46876; AAM46877; AAM46878; AAM46879; AAM46880;AAM46881; AAM46882; AAM46883; AAM46884; AAM46885; AAM46886; AAM46887;AAM46888; AAM46889; AAM46890; AAM46891; AAM82560; AAM82561; AAM82562;AAM88280; AAM88283; AAN01150; AAN01151; AAN01152; AAN01153; AAN01154;AAN01155; AAN01156; AAN01157; AAN01158; AAN01159; AAN01160; AAN01161;AAN01162; AAN01163; AAN01164; AAN01165; AAN01166; AAN01167; AAQ10355;AAQ10356; AAQ10357; AAQ10358; AAQ10359; AAQ10360; AAQ10361; AAQ10362;AAQ10363; AAQ10364; AAQ10365; AAQ10366; AAQ10370; AAQ10371; AAQ10374;AAQ10375; AAQ10376; AAQ10377; AAQ10378; AAQ10379; AAQ10381; AAQ10382;AAQ10383; AAQ10384; AAQ10397; AAQ10398; AAQ10399; AAQ10400; AAQ10401;AAQ10402; CAA11167; CAA11168; CAA11169; CAA11170; CAA11171; CAA11172;CAC81013; CAC81016; CAC81017; CAC81018; CAC40044; CAC40045; CAC40046;CAC40047; CAC40048; CAC40049; CAC40050; CAC40051; CAC36995; CAC37007;CAC37327; CAC86626; CAD20322; CAD20336; CAD44999; CAG27339; CAG27340;CAG27341; CAG27342; CAG28960; CAG28961; CAG28962; CAG34129; CAH56424;CAJ32551; CAJ32558; CAD22811; CAD22818; AAK53066; AAK62039; AAK62040;AAK62041; AAK62042; AAK62043; AAL06634; AAL06635; AAL06636; AAL06637;AAL06638; AAN17779; AAN63953; AAN63954; AAN63955; AAN63956; AAN63957;AAN63958; AAN83932; AAN83933; AAN83934; AAN83935; AAN83936; AAN83937;AAN83938; AAN83939; AAN83940; AAN83941; AAN83942; AAN83943; AAN83944;AAN83945; AAN83946; AAN83947; AAN83948; AAN83949; AAN83950; AAN83951;AAN83952; AAN83953; AAN83954; AAN83955; AAN83956; AAN83957; AAN83958;AAN83959; AAN83960; AAN83961; AAP21996; AAP21997; AAP23238; AAQ18434;AAQ18435; AAP79943; AAP79947; AAP79953; AAP79961; AAP79966; AAP79973;AAP79975; AAR12332; AAR12333; AAR12334; AAR12335; AAR12336; AAR12337;AAR12338; AAR12339; AAR12340; AAR12341; AAR12342; AAR12343; AAR12344;AAR12345; AAR12346; AAR12347; AAR12348; AAR12349; AAQ86988; AAQ85081;AAQ85082; AAQ85083; AAQ85084; AAQ85085; AAQ85086; AAQ85087; AAQ85088;AAQ85089; AAQ85090; AAQ85091; AAT12703; AAT12704; AAR90879; AAQ90291;AAQ92920; AAQ92921; AAQ92922; AAQ92923; AAQ92924; AAQ92925; AAQ92926;AAQ92927; AAQ92928; AAQ92929; AAQ92930; AAQ92931; AAR25201; AAR33033;AAT07998; AAT08000; AAT08002; AAT08004; AAT12654; AAT12655; AAT12656;AAT12657; AAT12658; AAT12659; AAT12660; AAT12661; AAT12662; AAT12663;AAT12664; AAT12665; AAT12666; AAT12667; AAT12668; AAT12669; AAT12670;AAT12671; AAT12672; AAT12673; AAT12674; AAT12675; AAT12676; AAS93870;AAS93871; AAS93872; AAS93873; AAS93874; AAS93875; AAS93876; AAS93877;AAS93878; AAS93879; AAS93880; AAS93881; AAS93882; AAS93883; AAS93884;AAT09637; AAT09638; AAT09639; AAT81341; AAT81342; AAT81343; AAT81344;AAT81345; AAT81346; AAT81347; AAT81348; AAT81349; AAT81350; AAT81351;AAT81352; AAT81353; AAT81354; AAT81355; AAT81356; AAT81357; AAT81358;AAT81359; AAT81360; AAT81361; AAT81362; AAU25861; AAU25871; AAT79527;AAT79528; AAT79529; AAT65319; AAT65321; AAT65324; AAT65333; AAT65334;AAT65345; AAT65349; AAT51806; AAT51807; AAT51808; AAT51809; AAT51810;AAT51811; AAT51812; AAT51813; AAT51814; AAT51815; AAT51816; AAT51817;AAT51818; AAT51819; AAT51820; AAT51821; AAT51822; AAT51823; AAT51824;AAT51825; AAT51826; AAT51827; AAT51828; AAT51829; AAT51830; AAT51831;AAT51832; AAT51833; AAT51834; AAT51835; AAT51836; AAT51837; AAT51838;AAT51839; AAT51840; AAT51841; AAT51842; AAT51843; AAT51844; AAT51845;AAT51846; AAT51847; AAT51848; AAT51849; AAT51850; AAT51851; AAT51852;AAT51853; AAT51854; AAT51855; AAT51856; AAT51857; AAT51858; AAT51859;AAT64666; AAT64667; AAT64668; AAT64669; AAT64670; AAT64671; AAT64672;AAT64673; AAT64674; AAT64675; AAT64676; AAT64677; AAT64678; AAT64679;AAT64680; AAT64681; AAT64682; AAT64683; AAT64684; AAT64685; AAT64686;AAT64687; AAT64688; AAT64689; AAT64690; AAT64691; AAT64692; AAT64693;AAT64694; AAT64695; AAT64696; AAT64697; AAT64698; AAT64699; AAT64700;AAT64701; AAT64702; AAT64703; AAT64704; AAT64705; AAT64706; AAT64707;AAT64708; AAT64709; AAT64710; AAT64711; AAT64712; AAT64713; AAT64714;AAT64715; AAT64716; AAT64717; AAT64718; AAT64719; AAT64720; AAT64721;AAT64722; AAT64723; AAT64724; AAT64725; AAT64726; AAT64727; AAT64728;AAT64729; AAT64730; AAT64731; AAT64732; AAT64733; AAT64734; AAT64735;AAT64736; AAT64737; AAT64738; AAT64739; AAT64740; AAT64741; AAT64742;AAT64743; AAT64744; AAT64745; AAT64746; AAT64747; AAT64748; AAT64749;AAT64750; AAT64751; AAT64752; AAT64753; AAT64754; AAT64755; AAT64756;AAT64757; AAT64758; AAT64759; AAT64760; AAT64761; AAT64762; AAT64763;AAT64764; AAT64765; AAT64766; AAT64767; AAT64768; AAT64769; AAT64770;AAT64771; AAT64772; AAT64773; AAT64774; AAT64775; AAT64776; AAT64777;AAT64778; AAT64779; AAT64780; AAT64781; AAT64782; AAT64783; AAT64784;AAT64785; AAT64786; AAT64787; AAT64788; AAT64789; AAT64790; AAT64791;AAT64792; AAT64793; AAT64794; AAT64795; AAT64796; AAT64797; AAT64798;AAT64799; AAT64800; AAT64801; AAT64802; AAT64803; AAT64804; AAT64805;AAT64806; AAT64807; AAT64808; AAT64809; AAT64810; AAT64811; AAT64812;AAT64813; AAT64814; AAT64815; AAT64816; AAT64817; AAT64818; AAT64819;AAT64820; AAT64821; AAT64822; AAT64823; AAT64824; AAT64825; AAT64826;AAT64827; AAT64828; AAT64829; AAT64830; AAT64831; AAT64832; AAT64833;AAT64834; AAT64835; AAT64836; AAT64837; AAT64838; AAT64839; AAT64840;AAT64841; AAT64842; AAT64843; AAT64844; AAT64845; AAT64846; AAT64847;AAT64848; AAT64849; AAT64850; AAT64851; AAT64852; AAT64853; AAT64854;AAT64855; AAT64856; AAT64857; AAT64858; AAT64859; AAT64860; AAT64861;AAT64862; AAT64863; AAT64864; AAT64865; AAT64866; AAT64867; AAT64868;AAT64869; AAT64870; AAT64871; AAT64872; AAT64873; AAT64874; AAT64875;AAT64876; AAT64877; AAT64878; AAT64879; AAT64880; AAT64881; AAT64882;AAT64883; AAT64884; AAT64885; AAT64886; AAU07825; AAU07826; AAU07827;AAU07828; AAU07829; AAU07830; AAU07831; AAU09399; AAW24444; AAW24445;AAW24446; AAW24447; AAW24448; AAW24449; AAW24450; AAW24451; AAU11522;AAU25949; AAV80797; AAV80798; AAW65986; AAW65987; AAW65988; AAW65989;AAW65990; AAW34374; AAW34375; AAW34376; AAW34377; AAW34378; AAW50838;AAW50839; AAW50840; AAW50841; AAX23575; AAW78047; AAW78048; AAW78049;AAW78050; AAW78051; AAW78052; AAX77666; AAX77667; AAX77668; AAX77669;AAX77670; AAX77671; AAX77672; AAX77673; AAX77674; AAX14851; AAX47732;AAX47733; AAX47734; AAX47735; AAX47736; AAX47737; AAX47738; AAX47739;AAX47740; AAX47741; AAX47742; AAX47743; AAX47744; AAX47745; AAX47746;AAX47747; AAX47748; AAX47749; AAX47750; AAX47751; AAX47752; AAX47753;AAX47754; AAX47755; AAX47756; AAX47757; AAX49559; AAX49562; AAY85891;AAY85892; AAY85893; AAY85894; AAY85895; AAY85896; AAY85897; AAY85898;AAY85899; AAY85900; AAY85901; AAY85902; AAY85903; AAY85904; AAY85905;AAY85906; AAY85907; AAY85908; AAY85909; AAY85910; AAY85911; AAX63815;AAX63816; AAX63817; AAX63818; AAX63819; AAX63820; AAX63821; AAX63822;AAX63823; AAX63824; AAX63825; AAX63826; AAX63827; AAX63828; AAY42043;AAY42044; AAY42045; AAY42046; AAY42047; AAY42048; AAY42049; AAY42050;AAY42051; AAY42052; AAY42053; AAY42054; AAY42055; AAY42056; AAY42057;AAY42058; AAY42059; AAY42060; AAY42061; AAY42062; AAY42063; AAY42064;AAY42065; AAY42066; AAY42067; AAX84524; AAX84525; AAX84526; AAX84527;AAX84528; AAX84529; AAX84530; AAX84531; AAX84532; AAX84533; AAX84534;AAX84535; AAX84536; AAX84537; AAX84538; AAX84539; AAX84540; AAX84541;AAX84542; AAX84543; AAX84544; AAX84545; AAX84546; AAX84547; AAX84548;CAL40875; AAX11455; AAX11475; AAX11485; AAX11495; AAX56420; AAX11505;AAY28295; AAX11515; AAX11565; AAX11575; AAX11585; AAX11595; AAX11605;AAX11615; AAX11635; AAX12731; AAX11465; AAY28571; AAX12751; AAX11525;AAX11535; AAX11545; AAX11555; AAX11625; AAX12741; AAX12761; AAX12771;AAX12781; AAX12791; AAX12801; AAX12811; AAX34061; AAX35821; AAX35831;AAX38237; AAX35841; AAX35851; AAX47525; AAX56490; AAX47515; AAX35861;AAX47535; AAX35871; AAX56380; AAX56390; AAX56400; AAX56410; AAX56430;AAX56440; AAX56450; AAX56460; AAX56470; AAX56480; AAX56500; AAX56510;AAX56520; AAX56540; AAX56550; AAX56560; AAX56570; AAX56580; AAX56590;AAX56600; AAX57644; AAX57654; AAX57664; AAX57674; AAX57684; AAX57694;AAX57704; AAX57714; AAX57733; AAX57734; AAX57744; AAX57754; AAX57764;AAX57774; AAX57784; AAX57794; AAX57804; AAX57814; AAX57824; AAX57834;AAX57844; AAX57854; AAX57864; AAX57874; AAX57884; AAX57894; AAX57904;AAX57914; AAX57924; AAX57934; AAX57944; AAX76623; AAX76633; AAX76643;AAX76653; AAX76663; AAY59035; AAX76673; AAX76683; AAX76693; AAX76703;AAX76713; AAY28375; AAX76723; AAX76733; AAX76743; AAX76753; AAX76763;AAY18086; AAY18096; AAY18611; AAY18585; AAY18106; AAY18116; AAY18564;AAY18126; AAY18136; AAY18146; AAY18156; AAY18166; AAY18176; AAY18186;AAY18196; AAY27863; AAY28385; AAY27843; AAY28561; AAY28345; AAY28395;AAY27853; AAY28325; AAY27959; AAY27994; AAY28004; AAY28014; AAY28265;AAY28275; AAY28285; AAY28363; AAY28648; AAY28305; AAY28315; AAY28335;AAY28355; AAY28638; AAY28405; AAY28486; AAY28521; AAY28531; AAY28541;AAY28628; AAY28551; AAY28618; AAY28581; AAY28591; AAY28608; AAY44610;AAY44906; AAY44620; AAY44621; AAY44631; AAY44896; AAY44641; AAY44796;AAY44795; AAY44785; AAY44775; AAY44765; AAY44755; AAY44651; AAY44661;AAY46371; AAY46381; AAY47013; AAY47023; AAY47052; AAY46391; AAY47075;AAY47085; AAY46416; AAY46426; AAY46436; AAY64192; AAY64202; AAY64212;AAY64252; AAY64272; AAY64292; AAY64222; AAY64312; AAY64232; AAY64242;AAY64322; AAY64262; AAY64282; AAY64342; AAY64302; AAY64352; AAY64332;AAY64392; AAY64362; AAY64372; AAY64382; AAY64402; AAY98770; AAY98037;AAY98047; AAY98187; AAY98057; AAY98067; AAY98077; AAY98087; AAY98097;AAY98107; AAY98117; AAY98127; AAY98137; AAY98147; AAY98157; AAY98167;AAY98177; AAY98195; AAY98217; AAY98207; AAY98237; AAY98227; AAY98247;AAY98319; AAY98329; AAY98339; AAY98353; AAY98366; AAY98376; AAY98386;AAY98396; AAY98406; AAZ38539; AAZ38561; AAZ38462; AAZ38473; AAZ38484;AAZ38495; AAZ38506; AAZ38517; AAZ38528; AAZ38583; AAZ38605; AAZ38550;AAZ38572; AAZ38594; AAZ38616; AAZ38638; AAZ38650; AAZ43370; AAZ43383;AAZ43394; AAZ43405; AAZ74386; AAZ74352; AAZ74363; AAZ74430; AAZ74397;AAZ74408; AAZ74419; AAZ74441; AAZ74452; AAZ74507; AAZ74463; AAZ74474;AAZ74485; AAZ74496; AAZ74529; AAZ74518; AAZ74540; AAZ74573; AAZ74606;AAZ74551; AAZ74595; AAZ74562; AAZ74584; AAZ74617; AAZ79505; AAZ79516;AAZ79527; AAZ79560; AAZ79571; AAZ79582; AAZ79626; AAZ79944; AAZ79593;AAZ79615; AAZ79627; AAZ79941; AAZ79963; AAZ79974; AAZ79985; AAZ80017;AAZ79996; AAZ80007; AAZ80030; AAZ83288; AAZ83242; AAZ83312; AAZ83266;AAZ83277; AAZ83323; AAZ83371; AAZ83382; AAZ83649; AAZ83688; ABA12740;ABA12751; ABA12762; ABA12780; ABA12773; ABA16214; ABA18048; ABA18134;ABA18156; ABA18026; ABA18123; ABA26799; ABA26700; ABA26711; ABA26722;ABA26733; ABA26744; ABA26755; ABA26766; ABA26777; ABA26788; ABA42269;ABA43167; ABA43178; ABA42291; ABA43336; ABA43200; ABA42302; ABA42313;ABA42335; ABA42346; ABA42989; ABA42357; ABA42368; ABA42379; ABA42390;ABA42401; ABA42412; ABA42443; ABA42454; ABA42465; ABA42476; ABA42487;ABA42498; ABA42978; ABA42939; ABA42928; ABA42509; ABA42520; ABA42531;ABA42542; ABA42553; ABA42564; ABA87242; ABA87253; ABB96509; ABB02836;ABB02847; ABB02858; ABB02869; ABB02880; ABB02891; ABB02902; ABB04283;ABB04294; ABB04305; ABB04316; ABB04327; ABB04338; ABB04349; ABB04360;ABB04371; ABB02947; ABB02958; ABB02969; ABB02980; ABB02991; ABB03002;ABB03013; ABB03024; ABB03035; ABB03046; ABB03057; ABB03068; ABB03079;ABB03090; ABB03112; ABB04906; ABB04917; ABB04928; ABB04939; ABB04950;ABB04961; ABB04983; ABB05183; ABB05194; ABB05205; ABB05216; ABB04994;ABB05005; ABB19704; ABB19712; ABB19723; ABB19744; ABB19758; ABB86785;ABB86796; ABB87034; ABB87377; ABB87388; ABB87399; ABB87410; ABB87421;ABB87429; ABB87440; ABB87451; ABB87462; ABB87789; ABB88149; ABB88150;ABB88152; ABB88162; ABB88173; ABB88183; ABB88256; ABB88309; ABB88342;ABB88369; ABB46547; ABB46392; ABB46403; ABB46414; ABB46425; ABB46436;ABB46447; ABB46458; ABB53614; ABB53625; ABB53652; ABB53663; ABB53674;ABB53685; ABB53696; ABB53718; ABB53751; ABB54514; ABB52376; ABB77853;ABB59996; ABB77864; ABC50167; ABC50178; ABC50189; ABB79716; ABB60007;ABB79731; ABB80034; ABB80023; ABB79788; ABB79799; ABB79810; ABB79957;ABB79968; ABB80001; ABB80012; ABB80137; ABB80148; ABB80159; ABB80081;ABB80092; ABB80185; ABB80196; ABB80207; ABB80229; ABB80492; ABB80503;ABB80514; ABB80748; ABB80218; ABB80529; ABB80641; ABB80661; ABB80240;ABB80251; ABB80672; ABB80682; ABB80693; ABB80704; ABB80737; ABB80715;ABB80724; ABB82183; ABB82227; ABB96319; ABB96330; ABB96341; ABB96352;ABB96363; ABB96374; ABB96395; ABB96498; ABB96520; ABB96531; ABC02234;ABC02288; ABC02299; ABC02332; ABC02321; ABC02255; ABC02266; ABC02310;ABC39805; ABC40642; ABC40544; ABC40555; ABC40608; ABC40619; ABC41692;ABC41703; ABC41725; ABC41952; ABC41736; ABC41953; ABC41964; ABC42014;ABC42114; ABC42125; ABC42136; ABC42147; ABC42929; ABC42940; ABC42158;ABC42169; ABC42180; ABC42192; ABC42951; ABC42307; ABC42318; ABC42346;ABC42461; ABC42962; ABC42494; ABC42505; ABC42516; ABC42527; ABC42973;ABC42984; ABC42995; ABC43006; ABC43017; ABC42574; ABC43028; ABC43039;ABC43050; ABC43061; ABC43072; ABC43083; ABC43094; ABC43105; ABC43116;ABC42585; ABC42596; ABC43127; ABC42607; ABC42618; ABC42629; ABC43138;ABC43149; ABC42640; ABC43160; ABC42651; ABC43171; ABC43182; ABC42662;ABC42673; ABC42684; ABC43475; ABC42695; ABC43486; ABC43497; ABC43508;ABC42706; ABC42717; ABC42728; ABC42739; ABC42761; ABC42772; ABC42783;ABC42794; ABC42805; ABC43519; ABC43530; ABC42816; ABC43541; ABC42827;ABC42838; ABC42849; ABC43552; ABC42860; ABC42871; ABC42882; ABC42893;ABC46554; ABC46565; ABC46576; ABC54668; ABC54679; ABC50200; ABC50211;ABC50222; ABC50233; ABC50244; ABC50255; ABC50266; ABC50277; ABC50288;ABC50299; ABC50310; ABC50321; ABC50332; ABC50343; ABC50354; ABC50365;ABC50376; ABC50387; ABC50398; ABC50409; ABC50420; ABC67319; ABC67817;ABC68233; ABC67850; ABC67883; ABC67894; ABC67967; ABC67978; ABC67454;ABC67989; ABC68000; ABC68049; ABC68060; ABC67471; ABC68071; ABC68093;ABC67543; ABC67554; ABC68082; ABC67565; ABC67576; ABC67587; ABC67598;ABC67609; ABC67620; ABC67631; ABC67642; ABC67653; ABC67664; ABC67675;ABC67686; ABC67697; ABC67708; ABC67719; ABC67733; ABC67806; ABC67828;ABC67839; ABC67861; ABC67872; ABC68222; ABC84389; ABC84400; ABC86148;ABC84411; ABC86124; ABC86040; ABC84422; ABC86029; ABC86018; ABC84433;ABC86007; ABC85996; ABC85985; ABC85974; ABC84444; ABC85963; ABC85952;ABD38134; ABC85941; ABC85930; ABC85919; ABC85908; ABC85897; ABC85886;ABC85875; ABC84498; ABC84509; ABC85864; ABC85853; ABC85842; ABC84520;ABC85831; ABC85765; ABC85820; ABC85809; ABC85798; ABC84531; ABC84542;ABC86137; ABC85787; ABC85776; ABC85754; ABC84560; ABC84571; ABD15526;ABD15537; ABD16538; ABD16527; ABD16516; ABD16560; ABD16505; ABD16494;ABD16483; ABD16472; ABD16358; ABD16347; ABD16336; ABD16325; ABD16314;ABD16303; ABD16549; ABD16762; ABD17334; ABD17323; ABC97374; ABD16751;ABD16740; ABD16729; ABD16718; ABD16593; ABD16582; ABD16571; ABD16292;ABD15790; ABD15779; ABD15768; ABD15757; ABD15746; ABD15735; ABD15724;ABD15713; ABD15702; ABD15691; ABD15680; ABD15669; ABD15658; ABD15647;ABD15504; ABD15493; ABD15482; ABD15471; ABD15460; ABD15449; ABD15625;ABD15614; ABD15603; ABD15592; ABD15581; ABD15570; ABD15559; ABD15548;ABD15636; ABD60790; ABD60801; ABD61293; ABD60812; ABD61304; ABD60823;ABD61529; ABD61757; ABD61777; ABD61315; ABD61326; ABE12078; ABD60834;ABD61337; ABD61348; ABD61359; ABD61370; ABD61381; ABD60845; ABD61392;ABD61403; ABD61260; ABD61271; ABD60922; ABD61282; ABD61551; ABD61249;ABE12532; ABE12623; ABD62833; ABD62794; ABD77598; ABD77609; ABD77620;ABD77631; ABD77642; ABD77653; ABD77664; ABD79123; ABD79134; ABD79145;ABD77686; ABE12645; ABD77697; ABD79156; ABD79167; ABD79178; ABD77741;ABD77752; ABD77763; ABD77774; ABD77785; ABD79032; ABD77829; ABD79189;ABD77840; ABD79200; ABD79211; ABD77851; ABD79222; ABD77862; ABD77873;ABD79233; ABD77884; ABD77895; ABD79244; ABD77906; ABD78049; ABD78115;ABD78126; ABD94734; ABD94745; ABD94767; ABD94822; ABD94833; ABD94844;ABD94855; ABD94866; ABD94877; ABD94888; ABD94899; ABD94910; ABD94921;ABD94932; ABD94954; ABE11911; ABE12123; ABE27164; ABE13076; ABE13323;ABE13471; ABE13555; ABE13595; ABE13606; ABE13617; ABE13628; ABE13639;ABE13652; ABE13824; ABE14019; ABE14030; ABE14041; ABE14052; ABE14063;ABE14124; ABE14464; ABE14840; ABE15578; ABF47550; ABF47594; ABF47616;ABF47858; ABF47902; ABI47947; ABI48006; ABF82651; ABF83447; ABF82695;ABF82706; ABG26758; ABG26769; ABG26802; ABG26846; ABG26857; ABG26868;ABG26879; ABG26890; ABG26901; ABG26912; ABG26923; ABG26934; ABG26956;ABG37131; ABG37142; ABG37153; ABG37164; ABG37175; ABG37186; ABG37197;ABG37208; ABG37219; ABG37230; ABG37241; ABG37252; ABG37263; ABG37274;ABG37285; ABG37296; ABG37307; ABG37318; ABG37329; ABG37340; ABG37351;ABG37373; ABG37406; ABG37417; ABG37428; ABG37439; ABG37450; ABG37461;ABG37472; ABG37483; ABG37494; ABG37505; ABG37516; ABG37527; ABG37538;ABG37549; ABG37560; ABG37571; ABG37582; ABG37593; ABG37604; ABG37615;ABG47851; ABG47862; ABG47873; ABG47884; ABG47895; ABG47906; ABG47917;ABG47928; ABG47939; ABG47950; ABG47961; ABG47972; ABG47983; ABG47994;ABG48005; ABG48016; ABG48027; ABG48038; ABG48060; ABG48071; ABG48082;ABG48093; ABG48104; ABG48115; ABG48126; ABG48137; ABG48148; ABG48159;ABG48170; ABG48181; ABG48192; ABG48203; ABG48214; ABG48225; ABG48236;ABG48247; ABG48258; ABG48269; ABG48280; ABG48291; ABG48302; ABG48313;ABG48324; ABG48335; ABG48346; ABG48357; ABG48368; ABG67135; ABG67146;ABG67667; ABG67157; ABG67168; ABG67179; ABG67190; ABG67201; ABG67212;ABG67223; ABG67234; ABG67245; ABG67502; ABG67513; ABG67524; ABG67535;ABG67546; ABG67557; ABG67568; ABG67579; ABG67590; ABG67601; ABG67612;ABG67623; ABG67634; ABG67645; ABG67656; ABG79941; ABG79963; ABG79974;ABG79985; ABG79996; ABG80007; ABG80018; ABG80029; ABG80040; ABG80051;ABG80062; ABG80073; ABG80084; ABG80095; ABG80106; ABG80117; ABG80128;ABG80139; ABG80150; ABG80161; ABG80194; ABG80205; ABG80216; ABG80227;ABG80238; ABG80249; ABG80260; ABG80271; ABG80282; ABG80293; ABG80304;ABG80315; ABG80326; ABG80337; ABG80348; ABG80359; ABG80370; ABG80381;ABG80392; ABG80403; ABG80414; ABG80425; ABG80436; ABG88289; ABG88355;ABG88366; ABG88377; ABG88388; ABG88399; ABG88410; ABG88421; ABG88432;ABG88443; ABG88454; ABG88465; ABG88476; ABG88487; ABG88498; ABG88509;ABG88520; ABG88531; ABG88564; ABG88575; ABG88586; ABG88597; ABG88608;ABG88619; ABG88630; ABG88641; ABG88652; ABG88663; ABG88674; ABG88685;ABG88696; ABG88707; ABG88718; ABG88729; ABG88740; ABG88751; ABG88762;ABG88773; ABG88784; ABG88795; ABG88806; ABG88817; ABI20793; ABI20815;ABI20881; ABI20892; ABI20903; ABI20914; ABI20925; ABI20936; ABI20947;ABI20958; ABI20969; ABI21736; ABI20980; ABI20991; ABI21002; ABI21013;ABI21024; ABI21035; ABI21046; ABI21057; ABI21068; ABI21079; ABI21090;ABI30444; ABI21101; ABI21112; ABI21123; ABI26646; ABI21134; ABI21145;ABI21156; ABI21167; ABI21178; ABI21244; ABI21255; ABI21266; ABI21277;ABI21288; ABI21299; ABI21310; ABI21321; ABI21332; ABI21343; ABI21354;ABI21365; ABI21376; ABI21387; ABI21398; ABI21409; ABI21420; ABI21431;ABI21442; ABI21453; ABI21464; ABI21475; ABI21486; ABI21497; ABI22159;ABI21508; ABI30389; ABI30400; ABI30411; ABI30422; ABI30433; ABI30455;ABI30466; ABI30477; ABI30488; ABI30499; ABI30510; ABI30521; ABI30532;ABI30543; ABI30554; ABI30576; ABI30587; ABI30598; ABI30609; ABI30620;ABI30733; ABI30744; ABI30755; ABI30766; ABI30777; ABI30788; ABI30799;ABI30810; ABI30821; ABI30832; ABI30843; ABI30854; ABI30865; ABI30876;ABI84400; ABI84412; ABI84471; ABI84486; ABI84577; ABI84806; ABI84938;ABI92258; ABI92269; ABI92280; ABI92291; ABI92324; ABI92335; ABI92346;ABI92357; ABI92368; ABI92390; ABI92401; ABI92412; ABI92423; ABI92434;ABI92445; ABI92456; ABI92467; ABI92478; ABI92489; ABI92500; ABI92511;ABI92522; ABI92533; ABI92544; ABI92555; ABI92566; ABI92577; ABI92588;ABI92599; ABI92610; ABI92621; ABI92632; ABI92643; ABI92654; ABI92665;ABI92676; ABI92687; ABI92698; ABI92709; ABI92720; ABI92731; ABI92742;ABI92753; ABI92764; ABI92775; ABI92786; ABI92797; ABI92808; ABI92819;ABI92830; ABI92841; ABI92852; ABI92863; ABI92874; ABI92885; ABI92896;ABI92907; ABI92918; ABI92929; ABI92940; ABI92951; ABI92962; ABI92973;ABI92984; ABI92995; ABI93006; ABI93017; ABI93039; ABI93050; ABI93061;ABI93072; ABI93083; ABI93094; ABI93105; ABI93116; ABI95474; ABK79937;ABI95228; ABI95239; ABI95305; ABJ09096; ABJ09107; ABJ09118; ABJ09140;ABJ09162; ABJ09173; ABJ09195; ABJ09206; ABJ09217; ABJ09228; ABJ09239;ABJ09250; ABJ09261; ABJ09272; ABJ09283; ABJ09294; ABJ09305; ABJ09316;ABJ09338; ABJ09349; ABJ09360; ABJ09371; ABJ09382; ABJ16587; ABJ16598;ABJ16620; ABJ16631; ABJ16697; ABJ16708; ABJ16741; ABJ16752; ABJ16763;ABJ16774; ABJ16785; ABJ53460; ABJ53471; ABJ53482; ABK39951; ABK39962;ABK39973; ABK39984; ABK40017; ABK40061; ABK40075; ABK40612; ABK40623;ABK40645; ABK40656; ABK40667; ABK40678; ABK40700; ABK40711; ABK40722;ABK40733; ABK40744; ABK79981; ABK79992; ABK80014; ABK80058; ABK80069;ABK80080; ABK80091; ABK80102; ABK80113; ABK80124; ABK80300; ABK80135;ABK80146; ABK80157; ABK80168; ABK80179; ABK80190; ABK80201; ABK80212;ABK80223; ABK80234; ABK80245; ABK80256; ABK80267; ABK80278; ABK80289;ABK80311; ABL67044; ABL67110; ABL67132; ABL67165; ABL67176; ABL67198;ABL67220; ABL67841; ABL67275; ABL67286; ABL67297; ABL67308; ABL67319;ABL67330; ABL67341; ABL67352; ABL67363; ABL67374; ABL67385; ABL67396;ABL67407; ABL67418; ABL75563; ABM21938; ABM22015; ABM22037; ABM22059;ABM22070; ABM22081; ABM22092; ABM22103; ABM22114; ABM22125; ABO32816;ABM22136; ABM22147; ABM22301; ABM22312; ABM22323; ABM22334; ABM22345;ABM22356; ABM22367; ABM66853; ABM66875; ABM66919; ABM66930; ABM66941;ABM66952; ABM66963; ABM66974; ABM66985; ABM66996; ABM67007; ABM67018;ABM67029; ABM67040; ABN50767; ABN50984; ABN50995; ABN51010; ABN51021;ABN51032; ABN51043; ABN51054; ABN51099; ABN51110; ABN51121; ABN51132;ABN51154; ABN59390; ABO32656; ABO32667; ABO32692; ABO32751; ABO32762;ABO32775; ABO32790; ABO32803; ABO32827; ABO32838; ABO32849; ABO32860;ABO32871; ABO32882; ABO32893; ABO32904; ABO32915; ABO32926; ABO32937;ABO33036; ABO33047; ABO33058; ABO33069; ABO38076; ABO38087; ABO38230;ABO38241; ABO38252; ABO38274; ABO38285; ABO38417; ABO44035; ABO44068;ABO44079; ABO51829; ABO51862; ABO51884; ABO76913; ABO52071; ABO52126;ABO52214; ABO52291; ABO52313; ABO52324; ABO52335; ABO52346; ABO52357;ABO52368; ABO52423; ABO52522; ABO52566; ABO52577; ABO52588; ABO52599;ABO52632; ABO52643; ABO52676; ABO64343; ABO76946; ABO76957; ABP49184;ABP49371; ABP49404; ABP49415; ABP49426; ABP49492; ABP49503; ABP49514;BAA00769; BAA00770; BAA00771; BAA00772; BAA01025; BAA01026; BAA02776;BAA02777; BAA02778; BAA02779; BAA04707; BAA04708; BAA04709; BAA04710;BAA04711; BAA04712; BAA04713; BAA04714; BAA04715; BAA04716; BAA04717;BAA04718; BAA04719; BAA21644; BAA21645; BAA21646; BAA21647; BAA21648;BAA33866; BAA33938; BAA33939; BAA33940; BAA33941; BAA33942; BAA33943;BAA33944; BAA33945; BAA33946; BAA33947; BAA07844; BAA07845; BAA07846;BAA07847; BAA07848; BAA07849; BAA07850; BAA08713; BAA08714; BAA21070;BAA08715; BAA08716; BAA08717; BAA08718; BAA21071; BAA08719; BAA13091;AAY23641; AAY23642; AAY25498; AAY46201; AAY46202; AAY58320; AAZ06795;AAZ32943; AAZ32944; AAZ32945; AAZ32946; AAZ32947; AAZ32948; AAZ32949;AAZ32950; AAZ32951; AAZ32952; AAZ32953; AAZ29151; AAZ29152; AAZ29153;AAZ29154; AAZ29155; AAZ29156; AAZ29157; AAZ29158; AAZ29159; AAZ29160;AAZ29161; AAZ29162; AAZ29163; AAZ29164; AAZ29165; AAZ29166; AAZ29167;AAZ29168; AAZ29169; AAZ29170; AAZ29171; AAZ29172; AAZ29173; AAZ29174;AAZ29175; AAZ29176; AAZ29177; AAZ29178; AAZ29179; AAZ29180; AAZ29181;AAZ29182; AAZ29183; AAZ29184; AAZ29185; AAZ29186; AAZ29187; AAZ29188;AAZ29189; AAZ29190; AAZ29191; AAZ29192; ABA39842; ABA39843; ABA39844;ABA39845; ABA39846; ABA39847; ABA39848; ABA39849; ABA39850; AAZ57437;AAZ74350; ABA46957; ABA41538; ABA27432; ABA27440; AAZ91962; AAZ91963;AAZ91964; ABA60253; ABA60254; ABA60255; ABA60256; ABA60257; ABA60258;ABA60259; ABA60260; ABA60261; ABA60262; ABA60263; ABA60264; ABA60265;ABA60266; ABA60267; ABA60268; ABA60269; ABA60270; ABA60271; ABA60272;ABA60273; ABA60274; ABA60275; ABA60276; ABA60277; ABA60278; ABA60279;ABA60280; ABA60281; ABA60282; ABA60283; ABA60284; ABA60285; ABA60286;ABA60287; ABA60288; ABA60289; ABA60290; ABA60291; ABA60292; ABA60293;ABA60294; ABA60295; ABA60296; ABA60297; ABA60298; ABA60299; ABA60300;ABA60301; ABA60302; ABA60303; ABA60304; ABA60305; ABA60306; ABA60307;ABA60308; ABA60309; ABA06580; ABA06581; ABA06582; ABA06583; ABA06584;ABA06585; ABA60900; ABA60901; ABA60902; ABA60903; ABA60904; ABA60905;ABA60906; ABA60907; ABA60908; ABA60909; ABA60910; ABA60911; ABA60912;ABA60913; ABA60914; ABA60915; ABA60916; ABA60917; ABA60918; ABA60919;ABA60920; ABA60921; ABA60922; ABA60923; ABA60924; ABA60925; ABA60926;ABA60927; ABA60928; ABA60929; ABA60930; ABA60931; ABA60932; ABA60933;ABA60934; ABA60935; ABA60936; ABA60937; ABA60938; ABA60939; ABA60940;ABA60941; ABA60942; ABA60943; ABA60944; ABA60945; ABA60946; ABA60947;ABA60948; ABA60949; ABA60950; ABA60951; ABA60952; ABA60953; ABA60954;ABA60955; ABA60956; ABA60957; ABA60958; ABA60959; ABA60960; ABA60961;ABA60962; ABA60963; ABA60964; ABA60965; ABA60966; ABA60967; ABA60968;ABA60969; ABA60970; ABA60971; ABA60972; ABA60973; ABA60974; ABA60975;ABA60976; ABA60977; ABA60978; ABA60979; ABA60980; ABA60981; ABA60982;ABA60983; ABA60984; ABA60985; ABA60986; ABA60987; ABA60988; ABA60989;ABA60990; ABA60991; ABA60992; ABA60993; ABA60994; ABA60995; ABA60996;ABA60997; ABA60998; ABA60999; ABA61000; ABA61001; ABA61002; ABA61003;ABA61004; ABA61005; ABA61006; ABA61007; ABA61008; ABA61009; ABA61010;ABA61011; ABA61012; ABA61013; ABA61014; ABA61015; ABA61016; ABA61017;ABA61018; ABA61019; ABA61020; ABA61021; ABA61022; ABA61023; ABA61024;ABA61025; ABA61026; ABA61027; ABA61028; ABA61029; ABA61030; ABA61031;ABA61032; ABA61033; ABA61034; ABA61035; ABA61036; ABA61037; ABA61038;ABA61039; ABA61040; ABA61041; ABA61042; ABA61043; ABA61044; ABA61045;ABB17173; ABB71825; ABB71826; ABB71827; ABB71828; ABB71829; ABB71830;ABB71831; ABB71832; ABB71833; ABB51981; ABB51982; ABB51983; ABB51961;ABB51963; ABB51964; ABB76697; ABB76698; ABB76699; ABB76700; ABB84191;ABB84192; ABB84193; ABB84194; ABB84195; ABB84196; ABB84197; ABB84198;ABB84199; ABB84200; ABB84201; ABB84202; ABC59709; ABC66247; ABC66248;ABC66249; ABC66250; ABC66251; ABC66252; ABC66253; ABC66254; ABC66255;ABC66256; ABC66257; ABC66258; ABD59850; ABD59851; ABD59852; ABD59853;ABD59854; ABD59855; ABD59856; ABD59857; ABD85122; ABF17954; ABF17955;ABF17956; ABF17957; ABF17958; ABF17959; ABE73114; ABE73115; ABF21268;ABF21269; ABF21271; ABF21273; ABF21281; ABG26247; ABG26248; ABG26249;ABG26250; ABG26251; ABG26252; ABG26253; ABG26254; ABG26255; ABG26256;ABG02860; ABG02861; ABG02862; ABG02863; ABH00993; ABH00994; ABH00995;ABH00996; ABH00997; ABH00998; ABH00999; ABH01000; ABH01001; ABH01002;ABH01003; ABH01004; ABH01005; ABH01006; ABH01007; ABH01008; ABH01009;ABH01010; ABH01011; ABH01012; ABH01013; ABH01014; ABH01015; ABH01016;ABH01017; ABH01018; ABH01019; ABH01020; ABH01021; ABH01022; ABI22056;ABI22057; ABI22058; ABI22059; ABI22060; ABI22061; ABI22062; ABI22063;ABI22064; ABI22065; ABI22066; ABI22067; ABI22068; ABI22069; ABI22070;ABI22071; ABI22072; ABI22073; ABI22074; ABI22075; ABI22076; ABI22077;ABI22078; ABI22079; ABI22080; ABI22081; ABI22082; ABI22083; ABI22084;ABI22085; ABI22086; ABI22087; ABI22088; ABI22089; ABI22090; ABI22091;ABI22092; ABI22093; ABI22094; ABI22095; ABI22096; ABI22097; ABI22098;ABI22099; ABI22100; ABI22101; ABI22102; ABI54388; ABI54389; ABJ51896;ABI49169; ABI49170; ABI49171; ABI49172; ABI49173; ABI49174; ABI49175;ABI49176; ABI49177; ABI49178; ABI49179; ABI49180; ABI49181; ABI49182;ABI49183; ABI49184; ABI51314; ABI51315; ABI51316; ABI55257; ABI55258;ABI55259; ABI55260; ABI55261; ABI55262; ABI55263; ABI55264; ABI55265;ABI55266; ABI55267; ABI55268; ABI55269; ABI55270; ABI55271; ABI55272;ABI55273; ABI55274; ABI55275; ABI55276; ABI55277; ABI55278; ABJ53158;ABM47075; ABK60213; ABK60214; ABK60215; ABL86145; ABM98421; ABM98422;ABO10165; ABO10166; ABO10167; ABO10168; ABO10169; ABO10170; ABO10171;ABO10172; ABO10173; ABO10174; ABO10175; ABO10182; ABO20947; ABO20952;ABO20953; ABO20954; ABO20955; ABO20956; ABO20957; ABO20958; ABO21717;ABO21718; ABO21719; ABO21720; ABO21721; ABO21722; ABO21726; ABO21727;ABO21728; ABO21729; ABO20959; ABO20960; ABO21733; ABO37477; ABO37478;ABO37479; ABO37480; ABO37481; ABO37482; ABO37483; ABO37484; ABO37485;ABO37486; ABO37487; ABO37488; ABO37489; ABO37490; ABO37491; ABO37492;ABO37493; ABO37494; ABO37495; ABO37496; ABO37497; ABO37498; ABO37499;ABO37500; ABO37501; ABO37502; ABO37503; ABO37504; ABO37505; ABO37506;ABO37507; ABO37508; ABO37509; ABO37510; ABO37511; ABO37512; ABO37513;ABO37514; ABO37515; ABO37516; ABO37517; ABO37518; ABO37519; ABO37520;ABO37522; ABO37523; ABO37524; ABO37525; ABO37526; ABO37527; ABO37528;ABO37529; ABO37530; ABO37531; ABO37532; ABO37533; ABO37534; ABO37535;ABO37536; ABO37537; ABO37538; ABO37539; ABO37540; ABO37541; ABO37542;ABO37543; ABO37544; ABO37545; ABO37546; ABO37547; ABO37548; ABO37549;ABO37550; ABO37551; ABO37552; ABO37553; ABO37554; ABO37555; ABO37556;ABO37557; ABO37558; ABO37559; ABO37560; ABO37561; ABO37562; ABO37563;ABO37564; ABO37565; ABO37566; ABO37567; ABO37568; ABO37569; ABO37570;ABO37571; ABO37572; ABO37573; ABO37574; ABO37575; ABO37576; ABO37577;ABO37578; ABO37579; ABO37580; ABO37581; ABO37582; ABO37583; ABO37584;ABO37585; ABO37586; ABO37587; ABO37588; ABO37589; ABO37590; ABO37591;ABO37592; ABO37593; ABO37594; ABO37595; ABO37596; ABO37597; ABO37598;ABO37599; ABO37600; ABO37601; ABO37602; ABO37603; ABO37604; ABO37605;ABO37606; ABO37607; ABO37608; ABO37609; ABO37610; ABO37611; ABO37612;ABO37613; ABO37614; ABO37615; ABO37616; ABO37617; ABO37618; ABO37619;ABO37620; ABO37621; ABO37622; ABO37623; ABO37624; ABO37625; ABO37626;ABO37627; ABO37628; ABO37629; ABO37630; ABO37631; ABO37632; ABO37633;ABO37634; ABO37635; ABO37636; ABO37637; ABO37638; ABO37639; ABO37640;ABO37641; ABO37642; ABO37643; ABO37644; ABO37645; ABO37646; ABO37647;ABO37648; ABO37649; ABO37650; ABO37651; ABO37652; ABO37653; ABO37654;ABO37655; ABO37656; ABO37657; ABO37658; ABO37659; ABO37660; ABO37661;ABO37662; ABO37663; ABO37664; ABO37665; ABO37666; ABO37667; ABO37668;ABO37669; ABO37670; ABO37671; ABO37672; ABO37673; ABO37674; ABO37675;ABO37676; ABO37677; ABO37678; ABO37679; ABO37680; ABO37681; ABO37682;ABO37683; ABO37684; ABO37685; ABO37686; ABO37687; ABO37688; ABO37689;ABO37690; ABO37691; ABO37692; ABO37693; ABO37694; ABO37695; ABO37696;ABO37697; ABO37698; ABO37699; ABO37700; ABO37701; ABO37702; ABO37703;ABO37704; ABO37705; ABO37706; ABO37707; ABO37708; ABO37709; ABO37710;ABO37711; ABO37712; ABO37713; ABO37714; ABO37715; ABO37716; ABO37717;ABO37718; ABO37719; ABO37720; ABO37721; ABO37722; ABO37723; ABO37724;ABO37725; ABO37726; ABO37727; ABO37728; ABO37729; ABO37730; ABO37731;ABO37732; ABO37733; ABO37734; ABO37735; ABO37736; ABO37737; ABO37738;ABO37739; ABO37740; ABO37741; ABO37742; ABO37743; ABO37744; ABO37745;ABO37746; ABO37747; ABO37748; ABO37749; ABO37750; ABO37751; ABO37752;ABO37753; ABO37754; ABO37755; ABO37756; ABO37757; ABO37758; ABO37759;ABO37760; ABO37761; ABO37762; ABO37763; ABO37764; ABO37765; ABO37766;ABO37768; ABO37769; ABO37770; ABO37771; ABO37772; ABO37773; ABO37774;ABO37775; ABO37776; ABO37777; ABO37778; ABO37779; ABO37780; ABO37781;ABO37782; ABO37783; ABP35587; ABP35588; ABP35589; ABP35590; ABP35591;ABP35592; ABP35593; ABP35594; ABP35595; ABP35596; ABP35597; ABP35598;ABP35599; ABP35600; ABP35601; ABP35602; AAA43178; AAA43182; AAA43187;AAA43200; AAA43184; AAA43195; AAA62328; AAA62335; AAA62329; AAA62331;AAA62327; AAA62330; AAA62339; AAA62338; AAA62332; AAA43230; AAA43229;AAA43228; AAA43227; AAA43226; ABG66978; ABE73717; ABG66979; ABG66980;ABG66981; ABG57281; ABG57282; ABG57283; ABG57284; AAA62470; AAA64229;AAA64228; AAB36975; AAB36976; AAB36977; AAB36978; AAB36979; AAB36980;AAB19009; AAB19010; AAB19011; AAB19012; AAB19013; AAB19014; AAB19015;AAB19016; AAB19017; AAB19018; AAB19019; AAB19020; AAB19021; AAB19022;AAB19023; AAB19024; AAB19025; AAB19028; AAB19026; AAB19027; AAA43143;AAA43144; AAA43145; AAA43146; AAA43147; AAA43148; AAA43149; AAA43211;AAA43212; AAA43275; AAA43114; AAA43105; AAA43111; AAA43100; AAA43107;AAA43101; AAA43109; AAA43110; AAA43112; AAA43102; AAA43103; AAO49821;AAA43163; AAA43164; AAA43099; AAA43239; AAA43155; AAA43156; AAA43162;AAA43165; AAA43151; ABF60581; ABF60577; ABF60576; ABF60580; ABF60579;ABF60578; AAB27733; AAB33340; AAA85781; AAA18781; AAA18782; AAC79579;AAA87553; AAB09413; AAB09414; AAB09415; AAB09416; AAB09417; AAB09418;AAB09419; AAB09420; AAB09421; AAA92927; AAB02560; AAD00123; AAD00124;AAC80152; AAD00125; AAD00126; AAC80153; AAD00127; AAD00128; AAC80154;AAF24003; AAC08288; AAC08289; AAC08290; AAC08291; AAC08292; AAC08293;AAC08294; AAC08295; AAC08296; AAC08297; AAB58297; CAA24269; CAA24270;CAA24271; CAA24273; CAA24281; CAA24290; CAA24291; CAA29337; CAA48482;CAA51904; CAA51905; CAA51906; CAA53437; CAA59412; CAA59413; CAA59414;CAA59415; CAA59416; CAA59417; CAA64893; CAA64894; CAA74382; CAA74383;CAA74384; CAA74385; CAA74386; CAA74387; CAA74388; CAA86526; CAA86527;CAA86528; CAA86529; CAA86530; CAA86531; CAA86532; CAA86533; CAA86534;CAA86535; CAA86536; CAA86537; CAA86538; CAA86539; CAA86540; CAA86541;CAA86542; CAA86543; CAA86544; CAA86545; CAA86546; CAA86547; CAA86548;CAA86549; CAA86550; CAA86551 and CAA86552.

Protein sequences of influenza virus subtype H4 hemagglutinin suitablefor inclusion as a cargo moiety in conjugates herein have the followingaccession numbers and are available to the public via the NationalCenter for Biotechnology Information (NCBI) Entrez protein search andretrieval system:

BAF43432; BAF43456; ABB87539; ABB87550; ABB87561; ABB87572; ABB87583;ABB87594; ABB87604; ABB87615; ABB87626; ABB87637; ABB87648; ABB87656;ABB87667; ABB87678; ABB87689; ABB87700; ABB88194; ABB88267; ABB88298;ABI47995; ABI48017; ABG88223; ABG88234; ABI92225; ABI84388; ABI84423;ABI84483; ABI84604; ABI84643; ABI84795; ABI84885; ABI84894; ABI84905;ABI92203; ABI92214; ABI97487; ABL67033; ABL67088; ABO51840; ABO51851;ABO51873; ABO51895; ABO51906; ABO51928; ABO51939; ABO51950; ABO52192;ABO52500; ABO52511; ABO52533; ABO52654; ABO52665; ABO52687; BAA14332;AAY88147; AAY88148; AAY88149; AAY88150; AAY88151; AAY88152; AAY88153;AAY88154; AAY88155; AAY88156; AAY88157; AAY88158; AAY88159; AAY88160;AAY88161; AAY88162; AAY88163; AAY88164; AAY88165; AAY88166; AAY88167;ABB80525; ABC59902; ABI17551; ABJ53168; AAA43179; AAA43216; AAA43217;AAA43218; AAA43219; AAA43220; AAA43221; AAA43222; AAA43223; AAA43224;BAF43458; BAF46756; BAF46758; BAF46904; BAF48476; BAF48478; AAG17427;AAG17429; AAF99711; CAD45001; CAD45000; AAN83962; AAN83963; AAN83964;AAN83965; AAN83966; AAN83967; AAN83968; AAN83969; AAN83970; AAN83971;AAT09640; AAT09641; AAT09642; AAT65318; AAT65320; AAT65322; AAT65335;AAT65336; AAT65338; AAT65346; AAT65347; ABB19802; ABB19847; ABB19867;ABB19878; ABB19886; ABB20362; ABB20372; ABB87473; ABB87484; ABB87495;ABB87506; ABB90165; ABB87517 and ABB87528.

Protein sequences of influenza virus subtype H5 hemagglutinin suitablefor inclusion as a cargo moiety in conjugates herein have the followingaccession numbers and are available to the public via the NationalCenter for Biotechnology Information (NCBI) Entrez protein search andretrieval system:

AB166862; AB188816; AB188824; AB189053; AB189061; AB212054; AB212280;AB212649; AB233319; AB233320; AB233321; AB233322; AB239125; AB241614;AB241615; AB241616; AB241617; AB241618; AB241619; AB241620; AB241621;AB241622; AB241623; AB241624; AB241625; AB241626; AB259712; AB261853;AB263192; AB263752; AB275420; AB275421; AB275422; AB275423; AB275424;AB275425; AB275426; AB275427; AB275428; AB275429; AB275430; AB275431;AB275432; AB275433; AB275434; AB284324; AB295603; AF028709; AF036356;AF046080; AF046088; AF046096; AF046097; AF046098; AF046099; AF046100;AF057291; AF082034; AF082035; AF082036; AF082037; AF082038; AF082039;AF082040; AF082041; AF082042; AF082043; AF084279; AF084280; AF084281;AF084532; AF098537; AF098538; AF098539; AF098540; AF098541; AF098542;AF098543; AF098544; AF098545; AF098546; AF100179; AF100180; AF102671;AF102672; AF102673; AF102674; AF102675; AF102676; AF102677; AF102678;AF102679; AF102680; AF102681; AF102682; AF144305; AF148678; AF164655;AF164656; AF164657; AF164658; AF164659; AF164660; AF164661; AF164662;AF164663; AF164664; AF164665; AF194169; AF194990; AF194991; AF194992;AF216713; AF216721; AF216729; AF216737; AF290443; AF303057; AF364334;AF377870; AF398417; AF398418; AF420254; AF439407; AF439408; AF468837;AF501234; AF501235; AF509016; AF509017; AF509018; AF509019; AF509020;AF509021; AF509022; AF509023; AF509024; AF509025; AF509026; AF509027;AF509028; AF509029; AF509030; AF509031; AF509032; AF509033; AF509034;AF509035; AF509036; AF509037; AF509038; AF509039; AJ305306; AJ621807;AJ621811; AJ632268; AJ632269; AJ715872; AJ867074; AJ971297; AJ971298;AJ972673; AM087222; AM183669; AM183670; AM183671; AM183672; AM183673;AM183674; AM183675; AM183676; AM183677; AM231714; AM236074; AM262541;AM262542; AM262543; AM262546; AM262547; AM262553; AM262572; AM397634;AM400972; AM400973; AM400974; AM400975; AM400976; AM400977; AM400978;AM400979; AM400980; AM400981; AM403460; AM403461; AM403462; AM403463;AM403464; AM403465; AM403466; AM403467; AM403468; AM403469; AM403470;AM403471; AM403472; AM403473; AM403474; AM403475; AM408209; AM408210;AM408211; AM408212; AM408213; AM408214; AM408215; AM408216; AM492165;AY059474; AY059475; AY059476; AY059477; AY059478; AY059479; AY059480;AY059481; AY059482; AY075027; AY075030; AY075033; AY221521; AY221522;AY221523; AY221524; AY221525; AY221526; AY221527; AY221528; AY221529;AY296064; AY296065; AY296066; AY296067; AY296068; AY296069; AY296070;AY296071; AY296072; AY296073; AY296074; AY296075; AY296076; AY296077;AY296078; AY296079; AY296080; AY296081; AY296082; AY296083; AY296084;AY296085; AY296086; AY497063; AY497064; AY497065; AY497066; AY497067;AY497068; AY497069; AY497070; AY497071; AY497072; AY497073; AY497074;AY497075; AY497076; AY497077; AY497078; AY497079; AY497080; AY497081;AY497082; AY497083; AY497084; AY497085; AY497086; AY497087; AY497088;AY497089; AY497090; AY497091; AY497092; AY497093; AY497094; AY497095;AY497096; AY500365; AY518362; AY526745; AY531029; AY534913; AY534914;AY535020; AY535021; AY535022; AY535023; AY536212; AY552000; AY552001;AY553784; AY553785; AY553786; AY553787; AY553788; AY553789; AY553790;AY553791; AY553792; AY553793; AY553794; AY553795; AY553796; AY553797;AY553798; AY553799; AY553800; AY553801; AY553802; AY553803; AY553804;AY553805; AY553806; AY553807; AY553808; AY553809; AY553810; AY553811;AY555150; AY555153; AY573917; AY574187; AY574190; AY575869; AY575870;AY575871; AY575872; AY575873; AY575874; AY575875; AY575876; AY575877;AY575878; AY575879; AY575880; AY576927; AY576930; AY577314; AY585357;AY585358; AY585359; AY585360; AY585361; AY585362; AY585363; AY585364;AY585365; AY585366; AY585367; AY585368; AY585369; AY585370; AY585371;AY585372; AY585373; AY585374; AY585375; AY585376; AY585377; AY590563;AY590568; AY590569; AY590570; AY590571; AY590572; AY590577; AY609312;AY623430; AY626143; AY627885; AY639405; AY646167; AY646175; AY646424;AY649382; AY651320; AY651321; AY651322; AY651323; AY651324; AY651325;AY651326; AY651327; AY651328; AY651329; AY651330; AY651331; AY651332;AY651333; AY651334; AY651335; AY651336; AY651337; AY651338; AY651339;AY651340; AY651341; AY651342; AY651343; AY651344; AY651345; AY651346;AY651347; AY651348; AY651349; AY651350; AY651351; AY651352; AY651353;AY651354; AY651355; AY651356; AY651357; AY651358; AY651359; AY651360;AY651361; AY651362; AY651363; AY651364; AY651365; AY651366; AY651367;AY651368; AY651369; AY651370; AY651371; AY651372; AY651373; AY653200;AY676033; AY676034; AY676035; AY676036; AY679514; AY684706; AY684894;AY720942; AY720945; AY720950; AY724783; AY724785; AY724787; AY724789;AY724791; AY724793; AY724795; AY728892; AY728894; AY737289; AY737296;AY737304; AY741213; AY741215; AY741217; AY741219; AY741221; AY747609;AY747617; AY770079; AY770991; AY779048; AY779050; AY786078; AY818135;AY818136; AY818137; AY830774; AY834279; AY842935; AY849793; AY854190;AY861372; AY866475; AY950230; AY950231; AY950232; AY950233; AY950234;AY950235; AY950236; AY972539; AY972540; AY972541; AY972542; AY995883;AY995884; AY995885; AY995886; AY995887; AY995888; AY995889; AY995890;AY995891; AY995892; AY995893; AY995894; AY995895; AY995896; AY995897;AY995898; CY005575; CY005918; CY005926; CY005927; CY005969; CY006028;CY006036; CY006040; CY011248; CY014168; CY014177; CY014185; CY014193;CY014197; CY014198; CY014199; CY014200; CY014201; CY14202; CY014203;CY014204; CY014205; CY014206; CY014207; CY014208; CY014209; CY014210;CY014211; CY014212; CY014213; CY014272; CY014280; CY014288; CY014296;CY014303; CY014311; CY014368; CY014376; CY014384; CY014393; CY014401;CY014409; CY014417; CY014425; CY014433; CY014441; CY014449; CY014457;CY014465; CY014477; CY014481; CY014489; CY014497; CY014510; CY014518;CY014529; CY014537; CY014543; CY014580; CY014615; CY014640; CY014642;CY014717; CY014722; CY014726; CY014849; CY014872; CY014984; CY015073;CY015081; CY015089; CY015115; CY016276; CY016284; CY016292; CY016300;CY016611; CY016779; CY016787; CY016795; CY016803; CY016811; CY016819;CY016827; CY016835; CY016843; CY016851; CY016859; CY016867; CY016875;CY016883; CY016891; CY016899; CY016907; CY016915; CY016923; CY016931;CY016939; CY16947; CY17027; CY017035; CY017043; CY017051; CY017059;CY017067; CY017179; CY017187; CY017403; CY017638; CY017646; CY017654;CY017662; CY017670; CY017678; CY017688; CY018949; CY019352; CY019360;CY019368; CY019376; CY019384; CY019392; CY019400; CY019408; CY019416;CY019424; CY019432; CY020229; CY020349; CY020621; CY020629; CY020637;CY020645; CY020653; CY020661; CY020669; CY020677; CY020693; CY020701;CY020709; CY021373; CY021381; CY021389; CY021397; CY021517; CY021525;DQ003215; DQ007623; DQ017270; DQ017271; DQ017272; DQ017273; DQ017274;DQ017275; DQ017276; DQ017277; DQ017278; DQ017279; DQ017280; DQ017281;DQ017282; DQ017283; DQ017284; DQ017285; DQ017286; DQ017287; DQ017288;DQ017289; DQ017290; DQ017291; DQ017292; DQ017293; DQ017294; DQ017295;DQ017296; DQ017297; DQ017298; DQ017299; DQ017300; DQ017301; DQ017302;DQ017303; DQ017304; DQ017305; DQ017306; DQ017307; DQ017308; DQ023145;DQ076201; DQ080022; DQ083550; DQ083551; DQ083552; DQ083553; DQ083554;DQ083555; DQ083556; DQ083557; DQ083558; DQ083559; DQ083560; DQ083561;DQ083562; DQ083563; DQ083564; DQ083565; DQ083566; DQ083567; DQ083568;DQ083569; DQ083570; DQ083571; DQ083572; DQ083573; DQ083574; DQ083575;DQ083576; DQ083577; DQ083578; DQ083579; DQ083580; DQ083581; DQ083582;DQ083583; DQ083584; DQ083585; DQ092869; DQ095612; DQ095613; DQ095614;DQ095615; DQ095616; DQ095617; DQ095618; DQ095619; DQ095620; DQ095621;DQ095622; DQ095623; DQ095624; DQ095625; DQ095626; DQ095627; DQ095628;DQ095629; DQ095630; DQ095631; DQ099755; DQ099756; DQ099757; DQ099758;DQ099759; DQ099760; DQ100554; DQ100555; DQ100556; DQ100557; DQ104701;DQ122147; DQ137873; DQ153251; DQ153252; DQ182483; DQ188905; DQ188906;DQ188907; DQ188908; DQ190857; DQ190858; DQ190859; DQ190860; DQ190861;DQ191688; DQ191689; DQ201829; DQ211922; DQ211923; DQ211924; DQ211925;DQ212792; DQ230521; DQ230522; DQ231240; DQ231241; DQ231242; DQ236077;DQ236085; DQ251447; DQ251796; DQ251797; DQ251798; DQ251799; DQ251800;DQ256383; DQ279301; DQ309440; DQ320137; DQ320875; DQ320876; DQ320877;DQ320878; DQ320879; DQ320880; DQ320881; DQ320882; DQ320883; DQ320884;DQ320885; DQ320886; DQ320887; DQ320888; DQ320889; DQ320890; DQ320891;DQ320892; DQ320893; DQ320894; DQ320895; DQ320896; DQ320897; DQ320898;DQ320899; DQ320900; DQ320901; DQ320902; DQ320903; DQ320904; DQ320905;DQ320906; DQ320907; DQ320908; DQ320909; DQ320910; DQ320911; DQ320912;DQ320913; DQ320914; DQ320915; DQ320916; DQ320917; DQ320918; DQ320919;DQ320920; DQ320921; DQ320922; DQ320923; DQ320924; DQ320925; DQ320926;DQ320927; DQ320928; DQ320929; DQ320930; DQ320931; DQ320932; DQ320933;DQ320934; DQ320935; DQ320936; DQ320937; DQ320938; DQ320939; DQ320940;DQ323672; DQ334760; DQ334768; DQ334776; DQ340848; DQ343150; DQ343151;DQ343152; DQ343502; DQ356886; DQ358746; DQ360835; DQ363918; DQ363923;DQ364996; DQ365004; DQ366306; DQ366314; DQ366322; DQ366330; DQ366338;DQ371928; DQ371929; DQ371930; DQ372591; DQ387854; DQ389158; DQ399540;DQ399547; DQ406728; DQ407519; DQ412997; DQ434889; DQ435200; DQ435201;DQ435202; DQ440535; DQ447199; DQ449031; DQ449632; DQ449640; DQ453141;DQ458992; DQ464354; DQ464377; DQ497642; DQ497643; DQ497644; DQ497645;DQ497646; DQ497647; DQ497648; DQ497649; DQ497650; DQ497651; DQ497652;DQ497653; DQ497654; DQ497655; DQ497656; DQ497657; DQ497658; DQ497659;DQ497660; DQ497661; DQ497662; DQ497663; DQ497664; DQ497665; DQ497666;DQ497667; DQ497668; DQ497669; DQ497670; DQ497671; DQ497672; DQ497673;DQ497674; DQ497675; DQ497676; DQ497677; DQ497678; DQ497679; DQ497680;DQ497681; DQ497682; DQ497683; DQ497684; DQ497685; DQ497686; DQ497687;DQ497688; DQ497689; DQ497690; DQ497691; DQ497692; DQ497693; DQ497694;DQ497695; DQ497696; DQ497697; DQ497698; DQ497699; DQ497700; DQ497701;DQ497702; DQ497703; DQ497704; DQ497705; DQ497706; DQ497707; DQ497708;DQ497709; DQ497710; DQ497711; DQ497712; DQ497713; DQ497714; DQ497715;DQ497716; DQ497717; DQ497718; DQ497719; DQ497720; DQ497721; DQ497722;DQ497723; DQ497724; DQ497725; DQ497726; DQ497727; DQ497728; DQ497729;DQ515984; DQ530173; DQ535724; DQ643809; DQ643982; DQ644955; DQ644956;DQ644957; DQ644958; DQ644959; DQ650659; DQ650663; DQ659113; DQ659326;DQ659327; DQ659679; DQ661910; DQ666146; DQ673901; DQ676830; DQ676834;DQ676838; DQ676840; DQ767725; DQ826532; DQ835313; DQ836043; DQ837587;DQ837588; DQ837589; DQ837590; DQ838508; DQ838509; DQ838516; DQ838517;DQ840519; DQ840533; DQ842487; DQ842489; DQ845348; DQ851561; DQ852600;DQ861291; DQ861999; DQ862000; DQ862001; DQ862002; DQ862003; DQ863503;DQ864711; DQ864715; DQ864716; DQ864717; DQ864718; DQ864719; DQ864720;DQ864721; DQ885610; DQ885612; DQ885614; DQ885616; DQ885618; DQ914808;DQ914814; DQ991231; DQ992714; DQ992715; DQ992716; DQ992717; DQ992718;DQ992719; DQ992720; DQ992721; DQ992722; DQ992723; DQ992724; DQ992725;DQ992726; DQ992727; DQ992728; DQ992729; DQ992730; DQ992731; DQ992732;DQ992733; DQ992734; DQ992735; DQ992736; DQ992737; DQ992738; DQ992739;DQ992740; DQ992741; DQ992742; DQ992743; DQ992744; DQ992745; DQ992746;DQ992747; DQ992748; DQ992749; DQ992750; DQ992751; DQ992752; DQ992753;DQ992754; DQ992755; DQ992756; DQ992757; DQ992758; DQ992759; DQ992760;DQ992761; DQ992762; DQ992763; DQ992764; DQ992765; DQ992766; DQ992767;DQ992768; DQ992769; DQ992770; DQ992771; DQ992772; DQ992773; DQ992774;DQ992775; DQ992776; DQ992777; DQ992778; DQ992779; DQ992780; DQ992781;DQ992782; DQ992783; DQ992784; DQ992785; DQ992786; DQ992787; DQ992788;DQ992789; DQ992790; DQ992791; DQ992792; DQ992793; DQ992794; DQ992795;DQ992796; DQ992797; DQ992798; DQ992799; DQ992800; DQ992801; DQ992802;DQ992803; DQ992804; DQ992805; DQ992806; DQ992807; DQ992808; DQ992809;DQ992810; DQ992811; DQ992812; DQ992813; DQ992814; DQ992815; DQ992816;DQ992817; DQ992818; DQ992819; DQ992820; DQ992821; DQ992822; DQ992823;DQ992824; DQ992825; DQ992826; DQ992827; DQ992828; DQ992829; DQ992830;DQ992831; DQ992832; DQ992833; DQ992834; DQ992835; DQ992836; DQ992837;DQ992838; DQ992839; DQ992840; DQ992841; DQ992842; DQ992843; DQ992844;DQ992845; DQ992846; DQ992847; DQ992848; DQ992849; DQ992850; DQ992851;DQ992852; DQ992853; DQ992854; DQ992855; DQ992856; DQ992857; DQ992858;DQ992859; DQ992860; DQ992861; DQ992862; DQ992863; DQ992864; DQ992865;DQ992866; DQ992867; DQ992868; DQ992869; DQ992870; DQ992871; DQ992872;DQ992873; DQ992874; DQ992875; DQ992876; DQ992877; DQ992878; DQ992879;DQ992880; DQ992881; DQ992882; DQ992883; DQ992884; DQ992885; DQ992886;DQ992887; DQ992888; DQ992889; DQ992890; DQ992891; DQ992892; DQ992893;DQ992894; DQ992895; DQ992896; DQ992897; DQ992898; DQ992899; DQ992900;DQ992901; DQ992902; DQ992903; DQ992904; DQ992905; DQ992906; DQ992907;DQ992908; DQ992909; DQ992910; DQ992911; DQ992912; DQ992913; DQ992914;DQ992915; DQ992916; DQ992917; DQ992918; DQ992919; DQ992920; DQ992921;DQ992922; DQ992923; DQ992924; DQ992925; DQ992926; DQ992927; DQ992928;DQ992929; DQ992930; DQ992931; DQ992932; DQ992933; DQ992934; DQ992935;DQ992936; DQ992937; DQ992938; DQ992939; DQ992940; DQ992941; DQ992942;DQ992943; DQ992944; DQ992945; DQ992946; DQ992947; DQ992948; DQ992949;DQ992950; DQ992951; DQ992952; DQ992953; DQ992954; DQ992955; DQ992956;DQ992957; DQ992958; DQ992959; DQ992960; DQ992961; DQ992962; DQ992963;DQ992964; DQ992965; DQ992966; DQ992967; DQ992968; DQ992969; DQ992970;DQ992971; DQ992972; DQ992973; DQ992974; DQ992975; DQ992976; DQ992977;DQ992978; DQ992979; DQ992980; DQ992981; DQ992982; DQ992983; DQ992984;DQ992985; DQ992986; DQ992987; DQ992988; DQ992989; DQ992990; DQ992991;DQ992992; DQ992993; DQ992994; DQ992995; DQ992996; DQ992997; DQ992998;DQ992999; DQ993000; DQ993001; DQ993002; DQ993003; DQ993004; DQ993005;DQ993006; DQ993007; DQ993008; DQ993009; DQ993010; DQ993011; DQ993012;DQ993013; DQ993014; DQ993015; DQ993016; DQ993017; DQ993018; DQ993019;DQ993020; DQ993021; DQ993022; DQ993023; DQ993024; DQ993025; DQ993026;DQ993027; DQ993028; DQ993029; DQ993030; DQ993031; DQ993032; DQ993033;DQ993034; DQ993035; DQ993036; DQ993037; DQ993038; DQ993039; DQ993040;DQ993041; DQ993042; DQ993043; DQ993044; DQ993045; DQ993046; DQ993047;DQ993048; DQ993049; DQ993050; DQ993051; DQ993052; DQ993053; DQ993054;DQ993055; DQ993056; DQ993057; DQ993058; DQ993059; DQ993060; DQ993061;DQ993062; DQ993063; DQ993064; DQ993065; DQ993066; DQ993067; DQ993068;DQ993069; DQ993070; DQ993071; DQ993072; DQ993073; DQ993074; DQ993075;DQ993076; DQ993077; DQ993078; DQ993079; DQ993080; DQ993081; DQ993082;DQ993083; DQ993084; DQ993085; DQ993086; DQ993087; DQ993088; DQ993089;DQ993090; DQ993091; DQ993092; DQ993093; DQ993094; DQ993095; DQ993096;DQ993097; DQ993098; DQ993099; DQ993100; DQ993101; DQ993102; DQ993103;DQ993104; DQ993105; DQ993106; DQ993107; DQ993108; DQ993109; DQ993110;DQ993111; DQ993112; DQ993113; DQ993114; DQ993115; DQ993116; DQ993117;DQ997076; DQ997087; DQ997094; DQ997102; DQ997111; DQ997122; DQ997123;DQ997133; DQ997163; DQ997182; DQ997218; DQ997219; DQ997253; DQ997262;DQ997268; DQ997276; DQ997283; DQ997308; DQ997325; DQ997352; DQ997355;DQ997361; DQ997370; DQ997377; DQ997392; DQ997396; DQ997405; DQ997410;DQ997513; DQ997522; DQ997531; DQ997538; DQ997547; DQ999872; DQ999880;DQ999887; EF041479; EF042614; EF042615; EF042616; EF042617; EF042618;EF042619; EF042620; EF042621; EF042622; EF042623; EF042624; EFO61116;EF090647; EF090648; EF090649; EF090650; EF107522; EF110518; EF110519;EF124794; EF165048; EF165049; EF165050; EF165051; EF165052; EF165053;EF165054; EF165055; EF165056; EF165057; EF165058; EF165059; EF165060;EF165061; EF165062; EF165063; EF165064; EF165065; EF165066; EF200512;EF200513; EF205154; EF205155; EF205156; EF205157; EF205158; EF205159;EF205160; EF382359; EF395844; EF395845; EF419242; EF419243; EF441263;EF441276; EF441277; EF441278; EF441279; EF441280; EF441281; EF446771;EF446779; EF447430; EF451059; EF456780; EF456781; EF456795; EF456798;EF456799; EF456802; EF456803; EF456805; EF467802; EF469650; EF469651;EF469652; EF469653; EF469654; EF469655; EF469656; EF469657; EF469658;EF469659; EF469660; EF473068; EF473069; EF473070; EF473073; EF473074;EF473075; EF473080; EF473081; EF474450; J02160; J04325; L46585; L46586;L46587; M10243; M18001; M18450; M18451; M30122; S68489; U05330; U05331;U05332; U20460; U20472; U20473; U20474; U20475; U28919; U28920; U37165;U37166; U37167; U37168; U37169; U37170; U37171; U37172; U37173; U37174;U37175; U37176; U37177; U37178; U37179; U37180; U37181; U37182; U67783;U69277; U79448; U79449; U79450; U79451; U79452; U79453; U79454; U79455;U79456; X07826 and X07869.

Protein sequences of influenza virus subtype H6 hemagglutinin suitablefor inclusion as a cargo moiety in conjugates herein have the followingaccession numbers and are available to the public via the NationalCenter for Biotechnology Information (NCBI) Entrez protein search andretrieval system:

BAF36386; BAF41914; CAC83641; CAC83642; CAC83644; CAC83645; CAC83646;CAC81274; CAC81276; CAC81279; CAC84237; CAC84238; CAC84239; CAC84240;CAC84241; CAC84242; CAC84243; CAC84244; CAD20327; CAD45192; CAD45193;CAD45194; CAD45195; CAD45196; CAD45197; CAD45198; CAG27343; CAG27344;CAG27345; CAG27346; CAG27347; AAT65326; AAT65328; AAT65330; AAT65332;AAT65340; AAT65341; AAT65342; AAT65343; AAT65344; AAT65350; AAV91218;AAX07773; AAV41833; AAW78053; AAX78820; ABB18391; ABB18402; ABB18476;ABB18951; ABB18962; ABB18973; ABB18978; ABB18994; ABB19011; ABB19020;ABB19026; ABB19032; ABB19042; ABB19055; ABB19072; ABB19083; ABB19094;ABB19101; ABB19107; ABB19118; ABB19129; ABB19140; ABB19151; ABB19162;ABB19173; ABB19184; ABB19195; ABB19206; ABB19217; ABB19228; ABB19239;ABB19360; ABB19371; ABB19382; ABB19393; ABB19404; ABB19585; ABB19596;ABB19947; ABB20283; ABB20294; ABB20387; ABB21783; ABO52005; ABG88267;ABI20804; ABI30356; ABI84387; ABI84457; ABI84466; ABI84473; ABI84516;ABI84663; ABI84827; ABI84838; ABI84866; ABI84916; ABI84927; ABI85172;ABI92192; ABI92236; ABI92247; ABI95151; ABI95162; ABI95173; ABI95184;ABI95195; ABJ16576; ABL67154; ABL75574; ABM21971; ABM21993; ABM22004;ABO51917; ABO51961; ABO51972; ABO51983; ABO51994; ABO52016; ABO52027;ABO52049; ABO52159; ABO52181; ABO52203; ABO52478; ABO52489; ABO76979;ABP49283; BAA14333; AAZ04680; AAZ04681; AAZ04682; AAZ04683; AAZ04684;AAZ04685; AAZ04686; AAZ04687; AAZ04688; AAZ04689; AAZ04690; AAZ04691;AAZ04692; AAZ04693; AAZ04694; AAZ04695; AAZ04696; AAZ04697; AAZ04698;AAZ04699; AAZ04700; AAZ04701; AAZ04702; AAZ04703; AAZ04704; AAZ04706;AAZ04707; AAZ04708; AAZ04709; AAZ04710; AAZ04711; AAZ04712; AAZ04713;AAZ04714; AAZ04715; ABB88830; ABD35522; ABD35523; ABD35524; ABD35525;ABD35526; ABD35527; ABD35528; ABD35529; ABD35530; ABD35531; ABD35532;ABD35533; ABD35534; ABD35535; ABD35536; ABD35537; ABD35538; ABD35539;ABD35540; ABD35541; ABD35542; ABD35543; ABD35544; ABD35545; ABD35546;ABD35547; ABD35548; ABD35549; ABD35550; ABD35551; ABD35552; ABD35553;ABD35554; ABD35555; ABD35556; ABD35557; ABD65973; ABD65981; ABD65988;ABH03489; ABH03497; AAA43198; BAF47393; BAF47395; BAF47399; BAF48480;BAF48639; BAF49413; AAF04721; AAF87507; AAG38550; AAG38551; AAG38552;AAM69944; AAM69945; AAM69946; AAM69947; AAM69948; AAM69949; AAM69950;AAM69951; AAM69962; AAM69973; AAM69983; AAM69993; AAM70005; AAM70007;AAO33479; AAO33480; AAO33481; AAO33482; AAO33483; AAO33484; AAO33485;AAO33486; AAO33487; AAO33488; CAC81746; CAC81747; CAC84981; CAC84982;CAC85087; CAC84852; CAC84860; CAC85080; CAC85081; CAC85082; CAC85083;CAC85084 and CAC85085.

Protein sequences of influenza virus subtype H7 hemagglutinin suitablefor inclusion as a cargo moiety in conjugates herein have the followingaccession numbers and are available to the public via the NationalCenter for Biotechnology Information (NCBI) Entrez protein search andretrieval system:

BAE96029; AAD26924; AAG10680; AAL37237; AAL37238; AAL37239; AAL37240;AAL37241; AAL37242; AAK58912; AAK58913; AAK58914; AAK58915; AAK58916;AAK58917; AAK58918; AAK58919; AAK58920; AAK58921; AAK58922; AAK58923;AAK58924; AAK58925; AAK58926; AAK58927; AAK58928; AAK58929; AAK58930;AAK58931; AAK58932; AAK58933; AAK58934; AAK58935; AAK58936; AAK58937;AAK58938; AAK58939; AAK58940; AAK58941; AAK58942; AAK58943; AAK58944;AAK58945; AAK58946; AAK58947; AAK58948; AAK58949; AAK58950; AAK58951;AAM19228; AAM19229; AAM19230; AAM19231; AAM19232; AAM19233; AAM19234;AAM19235; AAM19236; CAD33826; CAD37074; CAD38049; CAD38050; CAD38051;CAD38052; CAD38053; CAD38054; CAD38282; CAD38283; CAD38284; CAD38285;CAD38286; CAD38287; CAD38288; CAE45011; CAE48276; CAF04466; CAF33017;CAF33020; CAG27348; CAG27349; CAG28943; CAG28944; CAG28945; CAG28956;CAG28957; CAG28958; CAG28959; CAJ32548; CAJ32557; AAO86904; AAO86905;AAO86906; AAO86907; AAO86908; AAO86909; AAO86910; AAO86911; AAO86912;AAO86913; AAO86914; AAO86915; AAO86916; AAO86917; AAO86918; AAO86919;AAO86920; AAO86921; AAO86922; AAO86923; AAO86924; AAO86925; AAO86926;AAO86927; AAO86928; AAO86929; AAO86930; AAO86931; AAO86932; AAO86933;AAO86934; AAO86935; AAO86936; AAO86937; AAO86938; AAO86939; AAO86940;AAO86941; AAO86942; AAO86943; AAO86944; AAO86945; AAO86946; AAO86947;AAO86948; AAO86949; AAO86950; AAO86951; AAO86952; AAQ77402; AAQ77403;AAQ77404; AAQ77405; AAQ77406; AAQ77407; AAR02636; AAR02637; AAR02638;AAR02639; AAR02640; AAR02641; AAR02642; AAR02643; AAQ90292; AAS68158;AAT37403; AAT37404; AAT37405; AAT37406; AAT02538; AAT38819; AAT66415;AAT78582; AAT70170; AAT69348; AAV74187; AAU00821; AAU25838; AAU25943;AAU85295; AAU33999; AAU44367; AAU50675; AAV98693; AAV98694; AAV98695;AAY20940; AAY46207; AAY46208; AAY46209; AAY46210; AAY46211; AAY46212;AAY46213; AAY46214; AAY46215; AAY46216; AAY46217; AAY46218; AAY46219;AAY46220; AAY46221; ABB87303; ABB87751; ABB87762; ABB87773; ABB87784;ABB87800; ABB87822; ABB87833; ABB87854; ABB88289; ABB88359; ABI84433;ABI84462; ABI84599; ABI84602; ABI84683; ABI84694; ABI84849; ABI84981;ABI85000; ABI85011; ABI85029; ABI85038; ABI85084; ABI95206; ABM21982;ABO44145; ABO44156; ABO44167; ABO44178; ABO44189; ABO45248; ABO52060;ABO52698; ABO52709; ABO52764; ABO52775; ABO52786; ABO76990; ABO77001;ABO77012; ABO77056; ABO77067; ABO77078; ABO77089; ABP49206; ABP49228;AAY21164; AAY87433; AAY87443; ABF69256; ABG57088; ABG57089; ABG57090;ABG57091; ABG57092; ABG57093; ABH04379; ABH04385; ABH05673; ABI26074;ABI26075; ABJ90226; ABJ90237; ABJ90248; ABJ90259; ABJ90270; ABJ90280;ABO21714; ABO21715; AAA43192; AAR96248; AAA56803; AAA92244; AAA92245;AAA92246; AAA43152; AAA43154; AAA43150; AAA43237; AAA43087; AAA43174;AAC54376; AAC54377; AAC54379; AAC54380; AAC54381; AAC54382; AAC54383;AAC54384; AAC54385; AAC54386; AAC54387; AAC54388; AAC54389; CAA43815;CAA44429; CAA44430; CAA44431; CAA44432; CAA44433; CAA44434; CAA44435;CAA44436; CAA44437; CAA78263; CAA87393; BAE96040; BAE96041; BAE96042;BAE96043; BAE96044; BAE96045; BAF02913; BAF02930; BAF02931; BAF02932;BAF02933; BAF02934; BAF03206; BAF03525; BAF03526; BAF49200; BAF49202;BAF49411; AAC40998; AAC40999; AAD19847; AAD19848; AAD26922; AAD26923;AAD26925; AAD26926; AAD26927; AAD26928; AAD26929; AAD26930; AAD26931;AAD26932; AAD26933; AAD26934; AAD26935; AAD26936; AAD26937; AAD26938;AAD26939; AAD26940; AAD26941; AAD37422; AAG10650; AAG10651; AAG10652;AAG10653; AAG10654; AAG10655; AAG10656; AAG10657; AAG10658; AAG10659;AAG10660; AAG10661; AAG10662; AAG10663; AAG10664; AAG10665; AAG10666;AAG10667; AAG10668; AAG10669; AAG10670; AAG10671; AAG10672; AAG10673;AAG10674; AAG10675; AAG10676; AAG10677; AAG10678 and AAG10679.

Protein sequences of influenza virus subtype H8 hemagglutinin suitablefor inclusion as a cargo moiety in conjugates herein have the followingaccession numbers and are available to the public via the NationalCenter for Biotechnology Information (NCBI) Entrez protein search andretrieval system:

BAF43468; AAG38554; AAG38555; AAG38556; ABB87722; ABB87729; ABB87740;ABI84428; ABI84519; ABI85240; ABL67099; BAA14334; ABK32094 and AAA43177.

Protein sequences of influenza virus subtype H9 hemagglutinin suitablefor inclusion as a cargo moiety in conjugates herein have the followingaccession numbers and are available to the public via the NationalCenter for Biotechnology Information (NCBI) Entrez protein search andretrieval system:

AAA43208; AAD48995; AAD48996; AAD48997; AAD48998; AAD48999; AAD49000;AAF00701; AAF00702; AAF00703; AAF00704; AAF00705; AAF00706; AAF00707;AAF00708; AAF00709; AAF00710; AAF00711; AAF00712; AAF15580; AAF15581;AAF15582; AAF15583; AAF69255; AAF69256; AAF69257; AAF69258; AAF69259;AAF69260; AAF69261; AAF69262; AAG48164; AAG48165; AAG48166; AAG48167;AAG48168; AAG48169; AAG48170; AAG48171; AAG53035; AAG53036; AAG53037;AAG53038; AAG53039; AAG53040; AAG53041; AAG53042; AAG53043; AAG53044;AAG53045; AAG53046; AAG53047; AAG53048; AAG53049; AAG53050; AAG53051;AAG53052; AAG53053; AAG53054; AAG53055; AAG53056; AAG53057; AAG53058;AAG53059; AAG53060; AAG53061; AAG53062; AAG53063; AAG53064; AAG53065;AAG53066; AAG53067; AAG53068; AAG53069; AAK62979; AAK64189; AAL14080;AAL14081; AAL30486; AAL30487; AAL32475; AAL32476; AAL32477; AAL32478;AAL32479; AAL65235; AAL65236; AAL65237; AAL65238; AAL65239; AAL65240;AAL65241; AAL65242; AAL65243; AAL65244; AAL65245; AAL65246; AAL65247;AAL65248; AAL65249; AAL65250; AAL65251; AAL65252; AAL65253; AAL65254;AAL65255; AAL65256; AAL65257; AAL65258; AAM03341; AAM03342; AAN05676;AAN05677; AAN05678; AAN05679; AAN05680; AAN05681; AAN05682; AAN05683;AAN05684; AAN05685; AAN83972; AAN83973; AAN83974; AAN83975; AAN83976;AAN83977; AAN83978; AAN83979; AAN83980; AAN83981; AAN83982; AAN83983;AAN83984; AAN83985; AAN83986; AAN83987; AAO46077; AAO46078; AAO46079;AAO46080; AAO46081; AAO46082; AAO46083; AAO46084; AAO46085; AAO46086;AAO47744; AAO47745; AAO47746; AAO47747; AAO47748; AAO47749; AAO47750;AAO47751; AAO47752; AAP23303; AAP41031; AAP41032; AAP41033; AAP41034;AAP41035; AAP47821; AAP49029; AAP49030; AAP49031; AAP49032; AAP49033;AAP49034; AAP49035; AAP49036; AAP49037; AAP49038; AAP49039; AAP49040;AAP49041; AAP49042; AAP49043; AAP49044; AAP49045; AAP49046; AAP49047;AAP97867; AAQ04843; AAQ04844; AAQ04845; AAQ04846; AAQ04847; AAQ04848;AAQ04849; AAQ04850; AAQ04851; AAQ04852; AAQ04853; AAQ04854; AAQ04855;AAQ04856; AAQ04857; AAQ04858; AAQ04859; AAQ04860; AAQ04861; AAQ04862;AAQ04863; AAQ63104; AAQ63105; AAQ63106; AAQ63107; AAQ63108; AAQ63109;AAQ63110; AAQ63111; AAQ63112; AAQ63113; AAQ63114; AAQ63115; AAQ63116;AAQ63117; AAQ63118; AAQ63119; AAQ67246; AAQ97375; AAQ97376; AAQ97377;AAQ97378; AAQ97379; AAR08917; AAR08918; AAR98872; AAS48376; AAS48377;AAS48378; AAS48379; AAS48380; AAS48381; AAS48382; AAS48383; AAS48384;AAS48385; AAS48386; AAS48387; AAS48388; AAS48389; AAS48390; AAS48391;AAS48392; AAT12413; AAT37508; AAT45076; AAT65317; AAT65323; AAT65337;AAT65339; AAT70836; AAU00107; AAU00108; AAU00109; AAU11147; AAU11148;AAU11149; AAU11150; AAU11151; AAU11152; AAU11153; AAU11154; AAU11155;AAU11156; AAU11157; AAU11158; AAU11159; AAU11160; AAU11161; AAU11162;AAU11163; AAU11164; AAU11165; AAV30213; AAV52598; AAV52599; AAV52600;AAV52601; AAV52602; AAV52603; AAV52604; AAV52605; AAV67992; AAV68000;AAV68014; AAV68022; AAV68030; AAV68031; AAV68032; AAV68037; AAW29075;AAW29076; AAW29077; AAW29078; AAW29079; AAW29080; AAW50825; AAW50826;AAW78038; AAW78039; AAW78040; AAW78041; AAW78042; AAW78043; AAW78044;AAW78045; AAW78046; AAX32895; AAX32896; AAX51299; AAY27556; AAY52492;AAY52493; AAY52494; AAY52495; AAY52496; AAY52497; AAY52498; AAY52499;AAY52500; AAY52501; AAY52502; AAY52503; AAY52504; AAY52505; AAY52506;AAY52507; AAY52508; AAY52509; AAY52510; AAY52511; AAY52512; AAY52513;AAY52514; AAY52515; AAY52516; AAY52517; AAY52518; AAY52519; AAZ14102;AAZ14103; AAZ14104; AAZ14105; AAZ14106; AAZ14107; AAZ14108; AAZ14109;AAZ14110; AAZ14111; AAZ14112; AAZ14113; AAZ14114; AAZ14115; AAZ14116;AAZ14117; AAZ14118; AAZ14119; AAZ14120; AAZ14121; AAZ14122; AAZ14123;AAZ14124; AAZ14125; AAZ14126; AAZ14127; AAZ14128; AAZ14129; AAZ14977;AAZ14978; AAZ14979; AAZ14980; AAZ14981; AAZ14982; AAZ14983; AAZ14984;AAZ14985; AAZ14986; AAZ14987; AAZ14988; AAZ14989; AAZ14990; AAZ14991;AAZ14992; AAZ14993; AAZ14994; AAZ14995; AAZ14996; AAZ14997; AAZ14998;AAZ14999; AAZ15000; AAZ15001; AAZ15002; AAZ15003; AAZ15004; AAZ15005;AAZ15006; AAZ15007; AAZ15008; AAZ15009; AAZ15010; AAZ15011; AAZ15012;AAZ15013; AAZ15014; ABB03902; ABB17027; ABB17191; ABB19481; ABB19693;ABB20314; ABB20324; ABB20444; ABB51137; ABB58945; ABB58946; ABB58947;ABB58948; ABB58949; ABB58950; ABB58951; ABB58952; ABB58953; ABB58954;ABB58955; ABB87163; ABB87314; ABB87366; ABB87864; ABB87875; ABB87886;ABB87896; ABB87907; ABB87918; ABB87929; ABB87939; ABB87950; ABB88247;ABB88390; ABB90182; ABB90203; ABB90214; ABC48798; ABC48808; ABC48818;ABC48828; ABC48838; ABD61024; ABE02148; ABE27712; ABE27713; ABE27714;ABE27715; ABE27716; ABE27717; ABE27718; ABE28413; ABF56623; ABF56632;ABF56641; ABG27038; ABG27042; ABG27051; ABG27056; ABH12262; ABI17549;ABI17550; ABI84463; ABI84523; ABI94767; ABI94782; ABI96694; ABI96715;ABI96777; ABI97307; ABJ15706; ABK00113; ABK00119; ABK00143; ABK41621;ABK59023; ABM21875; ABM21876; ABM21877; ABM21878; ABM21879; ABM21880;ABM21881; ABM46227; ABM46228; ABM46229; ABM46230; ABM46231; ABM46232;ABM46233; ABM46234; ABM46235; ABM46236; ABM46237; ABM46238; ABM46239;ABM46240; ABM46241; ABM46242; ABM46243; ABM46244; ABM46245; ABM46246;ABM46247; ABM46248; ABM46249; ABM46250; ABM46251; ABM46252; ABM46253;ABM46254; ABM46255; ABM46256; ABM46257; ABM46258; ABM46259; ABM46260;ABM46261; ABM46262; ABM46263; ABM46264; ABM46265; ABM46266; ABM46267;ABM46268; ABM46269; ABM46270; ABM46271; ABM46272; ABM46273; ABM46274;ABM46275; ABM46276; ABM46277; ABM46278; ABM46279; ABM46280; ABM46281;ABM46282; ABM46283; ABM46284; ABM46285; ABM46286; ABM46287; ABM46288;ABM46289; ABM46290; ABM46291; ABM46292; ABM46293; ABM46294; ABM46295;ABM46296; ABM46297; ABM46298; ABM46299; BAA14335; BAB39511; BAB39512;BAB85614; BAB85615; BAB85616; BAB85617; BAB85618; BAD01514; BAD01515;BAD01516; BAD01517; BAD01518; BAE96033; BAF34373; BAF46427; BAF46437;BAF46447; BAF46457; BAF46467; BAF46477; BAF46487; BAF46497; BAF46507;BAF46517; BAF46527; BAF48357; CAB95856; CAB95857; CAC19694; CAD60401;CAD60402; CAD60403; CAH04111; CAH04112; CAH04113; CAH04114; CAH04115;CAH04116; CAH04117; CAH04118; CAH04119; CAH04120; CAJ32552; CAJ32553;CAL15444 and CAL15445.

Protein sequences of influenza virus subtype H10 hemagglutinin suitablefor inclusion as a cargo moiety in conjugates herein have the followingaccession numbers and are available to the public via the NationalCenter for Biotechnology Information (NCBI) Entrez protein search andretrieval system:

BAF03631; BAF31846; ABB87989; ABB88000; ABB88011; ABB88022; ABB88033;ABB88044; ABI84469; ABI84499; ABI84534; ABI84626; ABL67143; ABO52082;ABO52093; ABO52115; ABD23975; AAA43186; AAA79774; AAA79775; BAF43464;BAF46762; BAF46908; BAF47127; BAF48645; AAG33016; CAJ32549; CAJ32550;ABB87206; ABB87217; ABB87325; ABB87844; ABB87956; ABB87967 and ABB87978.

Protein sequences of influenza virus subtype H11 hemagglutinin suitablefor inclusion as a cargo moiety in conjugates herein have the followingaccession numbers and are available to the public via the NationalCenter for Biotechnology Information (NCBI) Entrez protein search andretrieval system:

BAF34926; BAF43435; ABD91535; ABD66294; ABD66295; ABD66296; ABD66297;ABD66298; ABF22671; ABM54148; AAA43188; AAA43191; AAA43183; AAA43203;AAA43181; BAF47125; BAF47129; BAF48643; BAF49417; AAG38553; AAV91221;ABB87228; ABB87239; ABB88055; ABB88066; ABB88077; ABB88088; ABI84440;ABI84442; ABI84545; ABI84556; ABI84600; ABI84723; ABJ53570; ABL67121;ABL67231; ABL75585; ABO52137; ABO52148; ABO52170; ABO52390; ABO52401;ABO52412; ABO52434; ABO52445; ABO52456; ABO52544; ABO52555; ABO76924;ABO76935; ABO76968; ABP49195; ABP49239; ABP49250; ABP49261; ABP49272;ABP49294; BAA14336; AAY85533; ABC59903; ABD91532; ABD91533 and ABD91534.

Protein sequences of influenza virus subtype H12 hemagglutinin suitablefor inclusion as a cargo moiety in conjugates herein have the followingaccession numbers and are available to the public via the NationalCenter for Biotechnology Information (NCBI) Entrez protein search andretrieval system:

BAF43416; BAF43433; AAG38557; AAG38558; AAG38559; CAL15446; ABB87195;ABB87249; ABB88099; ABB88110; ABB88121; ABG88278; ABI84446; ABI84489;ABJ09129; ABL67077; ABL67242; ABO52610; ABO52621; BAA14337; ABI17552 andAAA43180.

Protein sequences of influenza virus subtype H13 hemagglutinin suitablefor inclusion as a cargo moiety in conjugates herein have the followingaccession numbers and are available to the public via the NationalCenter for Biotechnology Information (NCBI) Entrez protein search andretrieval system:

BAF37821; BAF38383; BAF46906; CAJ32554; CAJ32555; AAV91212; AAV91213;ABB86511; ABB87334; ABB87345; ABB87811; ABI84452; ABI84566; ABI84601;BAA14338; ABG57285; AAA43213; AAA43214 and AAA43215.

Protein sequences of influenza virus subtype H14 hemagglutinin suitablefor inclusion as a cargo moiety in conjugates herein have the followingaccession numbers and are available to the public via the NationalCenter for Biotechnology Information (NCBI) Entrez protein search andretrieval system:

BAF43460 and ABI84453.

Protein sequences of influenza virus subtype H15 hemagglutinin suitablefor inclusion as a cargo moiety in conjugates herein have the followingaccession numbers and are available to the public via the NationalCenter for Biotechnology Information (NCBI) Entrez protein search andretrieval system:

ABB88138; ABB88320; ABB88331; AAA96134; ABB90704; BAF48363; ABB88132 andAAA92247.

Protein sequences of influenza virus subtype H16 hemagglutinin suitablefor inclusion as a cargo moiety in conjugates herein have the followingaccession numbers and are available to the public via the NationalCenter for Biotechnology Information (NCBI) Entrez protein search andretrieval system:

ABB87356; ABI84447; ABI85221; AAV91214; AAV91215; AAV91216 and AAV91217.

Polynucleotides encoding influenza virus subtype H1 hemagglutininssuitable for inclusion as a cargo moiety in conjugates herein have thefollowing accession numbers and are available to the public via theNational Center for Biotechnology Information (NCBI) Entrez nucleotidesearch and retrieval system:

AF222026; AF222034; AF222036; AF503481; AF503483; AY180460; AF534030;AF534045; AF534052; AY604795; AY604797; AY604798; AY604804; AY604806;CY004490; CY004539; CY006355; CY006779; CY009324; CY010092; CY010172;CY010228; CY010284; CY010324; CY010340; CY010996; CY011208; M59325;CY013565; CY013581; CY016563; CY017139; EF462556; EF462564; CY020173;CY021709; CY021749; CY021821; AB243744; AB255389; AB255390; AB255392;AB271115; X57491; AF222030; AF222031; AF222032; AF222033; AF503484;AF503485; AF503486; AF534048; AF534049; AY377936; AY604799; AY604800;AY604801; AY604802; AY604803; AY684125; DQ058215; CY003016; CY004498;CY005866; CY008148; CY009204; CY009276; CY009628; CY010108; CY010292;CY010300; CY010316; CY011012; CY011072; CY011088; CY011168; CY011184;CY011224; CY014627; CY014968; CY016052; CY016699; CY017123; CY017219;CY019763; CY019867; CY019883; EF467821; EF462563; X57493; Z46437;Z54288; L20111; L20113; L25072; U11858; L33780; L19022; L19016; L19014;M38312; L19006; L19024; K01331; U85986; AY029292; AF320059; AF320060;AF320062; AY129156; AF503475; AF503477; AY299509; AY297156; AY303741;AY701753; CY002672; CY002984; CY003688; CY003761; CY006419; DQ265706;DQ280203; CY009444; CY009868; CY009964; CY010556; CY010868; DQ666649;DQ666650; CY012440; DQ978382; DQ978389; DQ978390; EF462570; EF462571;CY020181; CY020861; CY021029; CY021693; CY021733; AB274304; Z46434;Z46435; Z46436; AJ289702; L19028; L19019; L19018; L19008; L19027;J02144; L19017; L19026; L24362; AY029288; AY029289; AF320063; AF503478;AF503479; AF503480; AY303747; CY000449; CY002360; CY002648; CY002688;CY003024; DQ249260; CY006107; CY003833; CY006667; CY006675; DQ280195;DQ280227; DQ280236; DQ280243; DQ280250; CY006867; CY009532; M34335;M33748; AF131993; AF131994; AF222027; AF222028; AF222035; AF503482;AF534027; AF534031; AF534038; AF534043; AF534044; AF534046; AF534053;AF534054; AY604796; AY604805; AY604807; AY604808; AY682833; CY002704;CY004504; CY004531; CY004546; CY005735; CY006363; CY003769; CY009220;CY009332; DQ397950; CY009452; DQ431990; CY010156; CY010164; CY010180;CY010236; CY010276; CY010332; M81707; CY011176; CY011192; CY011200;CY011216; CY015580; CY016308; CY016311; CY016313; CY016317; CY016318;CY016321; CY016327; CY16328; CY016331; CY016435; CY17003; CY017195;CY017235; CY17251; CY019101; CY020469; CY020485; ABO43478; ABO43483;ABO43484; ABO43491; ABO43493; ABO43494; X17221; J04572; J04574; L33480;L33482; L33487; L33489; L33490; L33755; L33743; L33745; L33747; L33753;U11703; U08903; U53162; S62154; S67220; U80948; U45451; U46943;AF305218; CY009756; CY009764; CY009780; CY009844; CY009956; CY010572;CY010740; CY010780; CY011952; DQ666648; CY013271; DQ981739; DQ978383;CY014733; CY015524; CY016228; CY016314; CY016315; CY016316; CY016324;CY016325; CY016326; CY016955; CY017011; CY017315; CY019085; CY020421;CY020437; CY020509; ABO43480; ABO43481; ABO43486; ABO43487; ABO43490;ABO43495; ABO43496; ABO43497; ABO43500; AB294217; L33483; L33484;L33485; L33486; L33492; L33493; CY011584; M59324; M59326; CY013573;CY013589; CY016643; CY018885; CY019205; CY019771; CY019875; CY019963;CY019971; EF462565; CY020917; CY021701; CY021717; CY021725; AB243745;AB255391; AB255393; AB255397; AB255398; AB271113; X57492; Z30276;Z54289; L20109; L20112; L20110; L25071; M73975; L19015; L19549; M38353;K01330; J02176; L19025; L19013; L19023; U03719; U03720; U72666;AY029291; AF320057; AF320058; AF320061; AF131995; AF250124; AF268312;AF268313; AF222029; AF534025; AF534026; AF534047; AF534050; AF534051;CY0001952; CY003288; CY004507; CY004592; CY009212; CY009596; DQ447187;CY010100; CY010116; CY010212; CY010220; CY010308; CY011004; CY011080;CY011152; CY011160; CY011232; M59327; M59328; CY012888; CY017115;CY017203; CY017211; CY017227; CY019739; CY019755; CY020189; CY021053;CY021757; CY021797; AB255394; AB255395; AB255396; X57494; X59778;AF398875; AF455679; AF455681; AF494246; AF494249; AF494250; AY289927;AY299499; AY299500; AY299502; CY002152; CY002616; CY002800; CY004466;CY004474; CY004482; DQ335992; DQ335995; CY009540; CY009612; CY009620;CY009796; CY009980; CY010356; CY010364; CY010380; CY010428; CY010444;CY010524; CY010540; DQ508897; DQ534416; CY011280; CY011296; CY011304;CY011392; CY012296; CY013821; CY013837; CY013853; CY013597; CY013871;CY016337; CY016338; CY016343; U02085; L09063; L33750; L33751; L33752;U37727; U38242; U53163; U80949; U46020; U46021; U46782; U46783; U46941;U46942; AF398878; AF455678; AY095226; AY095227; AF494251; AY289928;AY289929; AY633212; CY002536; CY002632; CY003000; DQ335998; CY009884;CY010372; CY009916; CY010388; CY010404; CY010460; CY010476; CY010492;CY010500; DQ508905; CY010764; CY011240; CY011312; CY012856; CY012864;CY012880; CY013032; CY015163; AF320067; AF503474; AF503476; AY299508;AY297157; AY303734; DQ139320; CY002352; CY002696; CY002992; CY003696;CY003704; DQ280212; DQ280219; CY006875; CY009772; CY009972; CY010804;CY010876; DQ666651; CY012824; CY013287; DQ978387; DQ978388; DQ978391;CY016196; CY016309; CY016310; CY016312; CY016319; CY016320; CY016322;CY016329; CY016330; CY020293; CY020453; CY020461; CY020477; ABO43479;ABO43482; ABO43485; ABO43492; V01088; X17224; J04573; CY016334;CY016335; CY016340; CY016341; CY016342; CY016347; CY016350; CY016357;CY016360; CY016361; CY016365; CY016366; CY016367; CY016370; CY017371;CY017869; CY019069; CY020237; CY020253; CY020261; U47310; AF026157;AF026160; AF091313; AF362778; AF362779; AF362784; AF362785; AF362786;AF362787; AF362794; AF362795; AF362796; AF362803; AF386775; AF386776;AF386777; AF386782; AF386783; AY038014; AY038338; AY038339; AY038344;AY038345; AY038346; L33481; L33488; L33491; L33756; L33744; L33746;L33754; L19005; K00992; U44482; U45452; AF398874; AF455680; AF494247;AF494248; AY299497; AY299498; AY299501; AY299503; CY002808; CY004458;CY006187; CY006195; DQ335993; DQ335994; CY009548; CY009604; CY009788;CY009804; CY009876; CY009892; CY010348; CY010412; CY010420; CY010436;CY010452; CY010508; CY010532; DQ508873; DQ508889; CY010772; DQ534415;DQ534417; DQ534418; CY011272; AY038347; AY038354; AY038355; AY038356;AY038357; AY060038; AY060044; AY233393; AY790289; AY851464; AY851465;AY851466; AY851467; AY971006; AY971007; AY971010; AY971011; DQ100426;DQ100427; CY002392; CY003296; CY003376; CY003392; CY003464; CY003480;CY006395; CY006403; DQ335999; DQ415318; CY009292; CY009316; CY009828;CY009940; CY010148; CY010204; CY010244; CY010828; CY010844; CY010884;CY010916; CY010924; CY010940; CY010956; CY011608; CY011776; CY016344;CY016346; CY016354; CY016363; CY016364; CY016371; CY016373; CY017363;CY017427; CY017435; CY019045; CY020269; CY021005; D00406; D00837;D00839; D10477; D29656; U47305; U47307; U96766; AF026153; AF026154;AF026156; AF055426; AF091309; AF091310; AF091312; AF362781; AF362783;AF362788; AF362791; AF362797; AF362798; AF362800; AF362802; AF386773;AF386774; AF386780; AY038335; AY038337; AY038341; AY038343; AY038351;AY038353; CY013829; CY013845; CY013879; CY0115167; CY016336; CY016339;CY016345; CY016352; CY016355; CY016362; CY016372; CY016459; CY017419;CY020277; D00407; D00838; D13570; D29657; U47304; U47306; U47308;AF026155; AF085413; AF117241; AF091308; AF091311; AF091317; AF362780;AF362782; AF362789; AF362790; AF362792; AF362799; AF362801; AF386779;AF386781; AF387491; AY038334; AY038336; AY038340; AY038342; AY038349;AY038350; AY038352; AY060031; AY038358; AF408859; AY060032; AY060039;AY060045; AY060047; AY060048; AY063229; AY184805; AY851463; AY851471;AY971003; AY971004; DQ118159; DQ118160; DQ118162; CY002528; CY003304;CY003328; CY003672; CY006171; CY006387; DQ336002; DQ336005; CY008524;CY008996; CY009172; CY009188; CY009228; CY009284; CY009340; CY010076;CY010132; CY010188; CY010268; CY010908; CY010964; CY010980; CY012608;CY011040; CY014007; DQ986134; CY016374; CY016380; CY016386; CY011792;CY013303; DQ978392; CY016244; CY016375; CY016376; CY016377; CY016382;CY016383; CY016391; CY016392; CY016393; DQ973300; CY019125; CY019221;CY019237; CY019923; CY020141; CY020157; CY020165; AB117167; AB117170;AB117171; AB117176; AB117177; AB117183; AB117192; AB117193; AB117203;AB117212; AB117213; AB117221; AB126622; AJ412708; AJ412709; AJ344013;AJ457868; AJ457869; AJ457878; AJ457879; AJ457884; AJ457885; AJ457886;AJ457894; AJ457895; AJ457904; AY060033; AY060034; AY060040; AY060046;AY060049; AY060050; AY063228; AF342821; AY590823; AY590824; AY790267;AY701755; AY851462; AY851469; AY851470; AY851472; AY861443; AY971005;DQ118158; DQ118161; DQ118163; CY003312; CY003320; CY003400; CY006427;CY006411; DQ336003; DQ336004; CY008988; DQ415316; CY009180; CY009196;CY009236; CY010196; CY010252; CY010260; CY010892; CY1900; CY010972;CY012800; CY016379; CY016381; CY016385; CY016387; CY016388; CY016389;CY016390; DQ973301; DQ973303; DQ973304; CY016971; CY017275; EF101749;CY017717; CY017725; CY017829; CY018933; CY019117; CY019779; CY019795;CY019803; CY020565; AB117166; AB117173; AB117174; AB117180; AB117182;AB117189; AB17190; AB117196; AB117199; AB117200; AB117202; AB117209;AB117210; AB17216; AB117219; AB117220; AB126630; AJ412712; AJ344002;AJ344019; AJ344020; AJ344009; AJ344010; AJ344012; AJ344022; AJ457865;AJ457872; AJ457875; AJ457881; CY016723; DQ973299; DQ973302; EF101741;CY017813; CY019109; CY020573; CY021629; AB117165; AB117172; AB117175;AB117181; AB117187; AB17188; AB17191; AB17197; AB17198; AB17201;AB17207; AB17208; AB117211; AB117217; AB117218; AJ412711; AJ344017;AJ344018; AJ344021; AJ344003; AJ344008; AJ344011; AJ457863; AJ457864;AJ457866; AJ457873; AJ457874; AJ457880; AJ457882; AJ457889; AJ457890;AJ457896; AJ457899; AJ457900; AJ457902; AJ457906; AJ457909; AJ457910;AJ457887; AJ457888; AJ457891; AJ457897; AJ457898; AJ457901; AJ457907;AJ457908; AJ457911; AJ489854; AJ489856; AJ517814; AJ517816; Z46441;Z54286; Z54287; L20106; L20117; L20116; L20108; L20107; U11857; L19021;L19011; L19020; L19012; U72667; U72668; U72669; AY029287; AY029290;AF320056; AF320064; AF320065; AF320066; AF503473; AY299506; AY299507;AY297154; AY297155; AY619961; CY002624; CY002640; CY002664; CY002680;CY003008; CY006747; CY006915; CY009860; CY010580; CY010852; DQ666644;DQ666645; DQ666646; DQ666647; CY013279; CY013295; DQ978384; DQ978385;DQ978386; CY014901; CY015443; CY015532; CY016260; CY016323; CY016963;CY017019; CY017243; CY019077; CY019093; CY019997; CY020429; CY020445;CY021037; ABO43488; ABO43489; ABO43498; ABO43499; U04857; U04858;U04859; L33757; L33758; L33748; L33749; U08904; U80950; AF455675;AF455676; AF455677; AF455682; AF389118; AY289930; AY282756; AY282757;AY299494; AY299495; AY299496; AY299504; AY299505; CY002568; CY002400;CY002656; DQ335991; DQ335996; DQ335997; CY009812; CY010396; CY010468;CY010484; DQ508857; DQ534419; CY011408; CY012304; CY012872; CY013040;CY013813; CY016332; CY016333; CY016348; CY016349; CY016358; CY016359;CY016368; CY016369; CY016691; CY017147; CY017155; CY017379; CY017877;CY019053; CY019061; CY019947; CY019955; CY020245; CY020285; D00840;D00841; D10163; D13571; D13572; D13573; D13574; D28518; D31949; U47309;AF026158; AF026159; AF085414; AJ457905; AJ489852; AJ489853; AJ489860;AJ489861; AJ489862; AJ517813; AJ517817; AJ517820; AJ489855; AJ489857;AJ489858; AJ517815; AF085415; AF085416; AF085417; AF116575; AF116576;AF091306; AF091307; AF091314; AF091315; AF091316; AF362793; AF386778;AY038333; AY038348; AY052778; AY060030; AY060035; AY060036; AY060037;AY060041; AY060042; AY060043; AY060051; AY060052; AY184806; AY590822;AY377929; AY851468; AY971008; AY971009; CY001680; DQ18164 and CY003368.

Polynucleotides encoding influenza virus subtype H2 hemagglutininssuitable for inclusion as a cargo moiety in conjugates herein have thefollowing accession numbers and are available to the public via theNational Center for Biotechnology Information (NCBI) Entrez nucleotidesearch and retrieval system:

AB056699; AB266382; AY209954; AY209955; AY209956; AY209957; AY209958;AY209959; AY209960; AY209961; AY209962; AY209963; AY209964; AY209965;AY209966; AY209967; AY209968; AY209969; AY209970; AY209971; AY209972;AY209973; AY209974; AY209975; AY209976; AY209977; AY209978; AY209979;AY209980; AY209981; AY209982; AY209983; AY209984; AY209985; AY209986;AY209987; AY209988; AY209989; AY422014; AY422015; AY422016; AY422017;AY633180; AY633196; AY633228; AY633364; AY633388; AY684893; CY003847;CY003855; CY003863; CY003871; CY003879; CY003887; CY003907; CY003914;CY003922; CY003936; CY003944; CY003952; CY003960; CY003968; CY003976;CY003984; CY003992; CY004554; CY005413; CY005538; CY005546; CY005765;CY005808; CY014556; CY014558; CY014601; CY014608; CY014609; CY014710;CY014821; CY014829; CY014976; CY015135; CY017693; CY018877; CY020317;CY020373; CY020381; CY020389; CY020397; CY020405; CY020413; CY020517;CY020541; CY020549; CY021013; CY021021; CY021069; CY021125; CY021789;CY021805; CY021813; D13575; D13576; D13577; D13578; D13579; D13580;DQ006282; DQ006283; DQ009917; DQ017486; DQ017493; DQ508841; DQ508881;J02127; J02154; L11125; L11126; L11127; L11128; L11129; L11130; L11131;L11132; L11133; L11134; L11135; L11136; L11137; L11138; L11139; L11140;L11141; L11142; L20406; L20407; L20408; L20409; L20410; AB275406;AB275414; AB275620; AB275628; AB275861; AB276115; AB292785; AB296074;AB298281; AF116197; AF116198; AF116199; AF116200; AF116201; AF16202;AF116203; AF16204; AF16205; AF16206; AF16207; AF16208; AF16209;AF116210; AF16211; AF231356; AF270716; AF270717; AF270718; AF270719;AF270720; AF270721; AF270722; AF270723; AF270724; AF270725; AF270726;AF270727; AF270728; AY180398; AY180399; AY180400; AY180401; AY180402;AY180403; AY209952 and AY209953.

Polynucleotides encoding influenza virus subtype H3 hemagglutininssuitable for inclusion as a cargo moiety in conjugates herein have thefollowing accession numbers and are available to the public via theNational Center for Biotechnology Information (NCBI) Entrez nucleotidesearch and retrieval system:

AB013806; AB013807; AB013808; AB013809; AB013810; AB013811; AB013812;AB013813; AB014060; AB014061; AB014062; AB019354; AB019355; AB019356;AB019357; AB043705; AB043706; AB043707; AB043708; AB221016; AB221017;AB221018; AB221019; AB221020; AB221021; AB221022; AB221023; AB221024;AB221025; AB221026; AB221027; AB221028; AB221029; AB221030; AB221031;AB221032; AB221033; AB221034; AB221035; AB243867; AB243868; AB243869;AB243870; AB243871; AB243872; AB243873; AB246366; AB259101; AB259102;AB259103; AB259104; AB259105; AB259106; AB259107; AB259108; AB259109;AB259110; AB259111; AB259112; AB259739; AB259740; AB259741; AB262301;AB262302; AB262303; AB262304; AB270992; AB270993; AB270994; AB270995;AB270996; AB270997; AB270998; AB270999; AB271000; AB271001; AB271002;AB271489; AB271490; AB271491; AB271492; AB271493; AB271494; AB271495;AB271496; AB271497; AB271498; AB271503; AB271504; AB271505; AB271506;AB271511; AB271512; AB271513; AB271514; AB271515; AB271516; AB271517;AB271524; AB271525; AB271526; AB271527; AB271528; AB271529; AB271530;AB271809; AB271810; AB271811; AB271812; AB271813; AB271814; AB271815;AB271816; AB271817; AB271818; AB271819; AB271820; AB271821; AB271822;AB271823; AB271824; AB271825; AB271826; AB271827; AB271828; AB271829;AB271830; AB271831; AB271832; AB271833; AB271834; AB271835; AB271836;AB271837; AB271838; AB271839; AB271840; AB271841; AB271842; AB271843;AB271844; AB271845; AB271846; AB271847; AB271848; AB271849; AB271850;AB275283; AB276113; AB277754; AB284320; AB289341; AB292402; AB292410;AB292660; AB292668; AB295605; AF008656; AF008657; AF008658; AF008659;AF008660; AF008661; AF008662; AF008663; AF008664; AF008665; AF008666;AF008667; AF008668; AF008669; AF008670; AF008671; AF008672; AF008673;AF008674; AF008675; AF008676; AF008677; AF008678; AF008679; AF008680;AF008681; AF008682; AF008683; AF008684; AF008685; AF008686; AF008687;AF008688; AF008689; AF008690; AF008691; AF008692; AF008693; AF008694;AF008695; AF008696; AF008697; AF008698; AF008699; AF008700; AF008701;AF008702; AF008703; AF008704; AF008705; AF008706; AF008707; AF008708;AF008709; AF008710; AF008711; AF008712; AF008713; AF008714; AF008715;AF008716; AF008717; AF008718; AF008719; AF008720; AF008721; AF008722;AF008723; AF008724; AF008725; AF008726; AF008727; AF008728; AF008729;AF008730; AF008731; AF008732; AF008733; AF008734; AF008735; AF008736;AF008737; AF008738; AF008739; AF008740; AF008741; AF008742; AF008743;AF008744; AF008745; AF008746; AF008747; AF008748; AF008749; AF008750;AF008751; AF008752; AF008753; AF008754; AF008755; AF008756; AF008757;AF008758; AF008759; AF008760; AF008761; AF008762; AF008763; AF008764;AF008765; AF008766; AF008767; AF008768; AF008769; AF008770; AF008771;AF008772; AF008773; AF008774; AF008775; AF008776; AF008777; AF008778;AF008779; AF008780; AF008781; AF008782; AF008783; AF008784; AF008785;AF008786; AF008787; AF008788; AF008789; AF008790; AF008791; AF008792;AF008793; AF008794; AF008795; AF008796; AF008797; AF008798; AF008799;AF008800; AF008801; AF008802; AF008803; AF008804; 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CY008172; CY008180; CY008196; CY008204; CY008212; CY008220;CY008228; CY008236; CY008244; CY008252; CY008260; CY008268; CY008276;CY008284; CY008292; CY008300; CY008308; CY008316; CY008324; CY008332;CY008340; CY008348; CY008356; CY008364; CY008372; CY008380; CY008388;CY008396; CY008404; CY008412; CY008420; CY008428; CY008436; CY008444;CY008452; CY008460; CY008468; CY008476; CY008484; CY008492; CY008500;CY008508; CY008516; CY008532; CY008540; CY008548; CY008556; CY008564;CY008572; CY008580; CY008588; CY008596; CY008604; CY008612; CY008620;CY008628; CY008636; CY008644; CY008652; CY008660; CY008668; CY008676;CY008684; CY008692; CY008700; CY008708; CY008716; CY008724; CY008732;CY008740; CY008748; CY008756; CY008764; CY008772; CY008780; CY008788;CY008796; CY008804; CY008812; CY008820; CY008828; CY008836; CY008844;CY008852; CY008860; CY008868; CY008876; CY008884; CY008892; CY008900;CY008908; CY008916; CY008924; CY008932; CY008940; CY008948; CY008956;CY008964; CY008972; CY008980; CY009004; CY009012; 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CY010716; CY010724; CY010732; CY010748; CY010756;CY010796; CY010812; CY010988; CY011020; CY011028; CY011048; CY011064;CY011120; CY011128; CY011136; CY011256; CY011264; CY011288; CY011320;CY011328; CY011336; CY011344; CY011352; CY011360; CY011368; CY011376;CY011384; CY011400; CY011416; CY011424; CY011432; CY011440; CY011448;CY01456; CY0111464; CY011472; CY011480; CY011488; CY011496; CY011504;CY011512; CY011520; CY011528; CY0111536; CY011544; CY011552; CY011560;CY011568; CY011576; CY011592; CY011616; CY011624; CY0111632; CY011640;CY011648; CY011656; CY011664; CY011672; CY011680; CY011688; CY011696;CY011704; CY011712; CY011720; CY011728; CY011736; CY011744; CY011752;CY011760; CY01768; CY0111808; CY011816; CY011824; CY011832; CY011840;CY011848; CY011856; CY011864; CY01872; CY0111880; CY011888; CY011896;CY011904; CY011912; CY011920; CY011928; CY011936; CY011944; CY011960;CY011968; CY011976; CY011984; CY011992; CY012000; CY012008; CY012016;CY012024; CY012032; CY012040; CY012048; CY012056; CY012064; CY012072;CY012080; CY012088; CY012096; CY012104; CY012112; CY012120; CY012128;CY012136; CY012144; CY012152; CY012160; CY012168; CY12176; CY012184;CY012200; CY012208; CY12216; CY12224; CY12232; CY12240; CY12248;CY12256; CY012264; CY012272; CY012280; CY012288; CY012312; CY012320;CY012328; CY012336; CY012344; CY012352; CY012360; CY012368; CY012376;CY012384; CY012392; CY012400; CY012408; CY012416; CY012424; CY012432;CY012448; CY012456; CY012464; CY012472; CY012480; CY012488; CY012496;CY012504; CY012512; CY012520; CY012528; CY012536; CY012544; CY012552;CY012560; CY12568; CY012576; CY012584; CY012592; CY012616; CY012624;CY012632; CY012640; CY012648; CY012656; CY012664; CY012672; CY012680;CY012688; CY012696; CY012704; CY012712; CY012720; CY012728; CY012736;CY012744; CY012752; CY012760; CY012768; CY012776; CY012784; CY012792;CY012848; CY012896; CY012904; CY012912; CY012920; CY012928; CY012936;CY012944; CY012952; CY012960; CY012968; CY012976; CY012984; CY012992;CY013000; CY013008; CY013016; CY013024; CY013048; CY013056; CY013064;CY013072; CY013080; CY013088; CY013096; CY013104; CY013112; CY013120;CY013128; CY013136; CY013144; CY13152; CY13160; CY13168; CY13176;CY13184; CY13192; CY013200; CY013208; CY013216; CY013224; CY013232;CY013240; CY013263; CY013311; CY013319; CY013327; CY013335; CY013343;CY013351; CY013359; CY013367; CY013375; CY013383; CY13389; CY013397;CY013405; CY013413; CY013421; CY013429; CY013437; CY013445; CY13453;CY013461; CY013469; CY013477; CY13485; CY13493; CY013501; CY13509;CY13517; CY13525; CY13533; CY013541; CY013549; CY013605; CY013613;CY013621; CY013629; CY013637; CY013645; CY13653; CY013661; CY013669;CY013677; CY013685; CY013693; CY013701; CY013709; CY013717; CY13725;CY013733; CY013741; CY013749; CY013757; CY013765; CY013773; CY013781;CY013789; CY013797; CY013805; CY013887; CY013895; CY013903; CY013911;CY013919; CY013927; CY013935; CY13943; CY013951; CY013959; CY013967;CY013975; CY013983; CY013991; CY013999; CY14015; CY14023; CY014031;CY014039; CY014047; CY014055; CY014063; CY014071; CY014079; CY014087;CY014095; CY014103; CY014111; CY14119; CY14127; CY14135; CY14143;CY014151; CY14159; CY14548; CY014571; CY014621; CY14633; CY14702;CY14865; CY014961; CY15492; CY15500; CY15508; CY015516; CY015540;CY015548; CY015556; CY015564; CY015572; CY015588; CY015596; CY015604;CY015612; CY015620; CY015628; CY015636; CY015644; CY015652; CY015660;CY015668; CY015676; CY015684; CY015692; CY015700; CY015708; CY015716;CY015724; CY015732; CY015740; CY015748; CY015756; CY015764; CY015772;CY015780; CY015788; CY015796; CY015804; CY015812; CY015820; CY015828;CY015836; CY015844; CY015852; CY015860; CY015868; CY015876; CY015884;CY015892; CY015900; CY015908; CY015916; CY015924; CY015932; CY015940;CY015948; CY015956; CY015964; CY015972; CY015980; CY015988; CY015996;CY016004; CY016012; CY016020; CY016028; CY016036; CY016044; CY016060;CY016068; CY016076; CY016084; CY016092; CY016100; CY016108; CY016116;CY016140; CY016180; CY016204; CY016212; CY016268; CY016395; CY016403;CY016411; CY016427; CY016443; CY016451; CY016467; CY016475; CY016483;CY016491; CY016499; CY016507; CY016515; CY016523; CY016531; CY016539;CY016547; CY016555; CY016571; CY016579; CY016587; CY016595; CY016603;CY016627; CY016635; CY016651; CY016659; CY016707; CY016715; CY016739;CY016747; CY016755; CY016763; CY016771; CY016979; CY016987; CY016995;CY017083; CY017091; CY017099; CY017107; CY017131; CY017163; CY017171;CY017259; CY017267; CY017283; CY017291; CY017299; CY017307; CY017323;CY017331; CY017339; CY017347; CY017355; CY017387; CY017395; CY017411;CY017443; CY017451; CY017459; CY017467; CY017475; CY017483; CY017491;CY017499; CY17507; CY017515; CY017523; CY017531; CY017539; CY017547;CY017555; CY017563; CY017571; CY17579; CY017587; CY017595; CY017603;CY017611; CY017619; CY017627; CY017709; CY017757; CY017773; CY017797;CY017805; CY017821; CY017837; CY017861; CY017885; CY017893; CY017901;CY17909; CY017917; CY017925; CY017933; CY017941; CY017949; CY017957;CY017965; CY017973; CY017981; CY017989; CY017999; CY18869; CY18925;CY018941; CY18957; CY185; CY18973; CY018981; CY018989; CY018997;CY019005; CY019013; CY019021; CY019029; CY019141; CY019149; CY019157;CY019165; CY019173; CY019181; CY019189; CY019197; CY019213; CY019245;CY019253; CY019261; CY019269; CY019277; CY019285; CY019293; CY019301;CY019309; CY019317; CY019325; CY019333; CY019747; CY019811; CY019819;CY019827; CY019835; CY019843; CY019851; CY19859; CY019891; CY019899;CY019907; CY019915; CY019931; CY019939; CY019981; CY019989; CY020005;CY020013; CY020021; CY020029; CY020037; CY020045; CY020053; CY020061;CY020069; CY020077; CY020085; CY020093; CY02101; CY020109; CY02117;CY020125; CY020133; CY020197; CY020205; CY020213; CY020221; CY020301;CY020309; CY020325; CY020333; CY020341; CY020357; CY020365; CY020493;CY020501; CY020525; CY020533; CY020717; CY020741; CY020757; CY020877;CY020893; CY020933; CY020997; CY021061; CY021077; CY021085; CY021093;CY021101; CY021109; CY021117; CY021157; CY021229; CY021261; CY021269;CY021277; CY021285; CY021309; CY021317; CY021341; CY021429; CY021453;CY021461; CY021597; CY021741; CY021765; CY021773; CY021781; CY021829;CY021837; CY021845; D00929; D00930; D00931; D00932; D10161; D10162;D13581; D13582; D13583; D13584; D21171; D21172; D21173; D21174; D21175;D21176; D21177; D21178; D21179; D21180; D21181; D21182; D21183; D30662;D30663; D30664; D30665; D30668; D30669; D30677; D30678; D30679; D30680;D30681; D30682; D30683; D30684; D30685; D30686; D43786; D43787; D43788;D43789; D43790; D43791; D43792; D49959; D49960; D49961; D49962; D49963;D49964; D49965; D49966; D49967; D86469; DQ006284; DQ006285; DQ007622;DQ021910; DQ021911; DQ059385; DQ066936; DQ086157; DQ086158; DQ086159;DQ086160; DQ086161; DQ089634; DQ089635; DQ089636; DQ089637; DQ089638;DQ089639; DQ114496; DQ114497; DQ114498; DQ114499; DQ114500; DQ114501;DQ114502; DQ114503; DQ114504; DQ114505; DQ114506; DQ114507; DQ114508;DQ114509; DQ114510; DQ114511; DQ114512; DQ114513; DQ114514; DQ114515;DQ114516; DQ114517; DQ114518; DQ114519; DQ114520; DQ114521; DQ114522;DQ114523; DQ114524; DQ114525; DQ114526; DQ114527; DQ114528; DQ114529;DQ114530; DQ114531; DQ114532; DQ114533; DQ114534; DQ114535; DQ114536;DQ114537; DQ124157; DQ124189; DQ124190; DQ124191; DQ124192; DQ124193;DQ124194; DQ124195; DQ124196; DQ132433; DQ141307; DQ145537; DQ146419;DQ150425; DQ150433; DQ159065; DQ159066; DQ159067; DQ167251; DQ167252;DQ167253; DQ167254; DQ167255; DQ167256; DQ167257; DQ167258; DQ167259;DQ167260; DQ167261; DQ167262; DQ167263; DQ167264; DQ167265; DQ167266;DQ167267; DQ167268; DQ167269; DQ167270; DQ167271; DQ167272; DQ167273;DQ167274; DQ167275; DQ167276; DQ167277; DQ167278; DQ167279; DQ167280;DQ167281; DQ167282; DQ167283; DQ167284; DQ167285; DQ167286; DQ167287;DQ167288; DQ167289; DQ167290; DQ167291; DQ167292; DQ167293; DQ167294;DQ167295; DQ167296; DQ167297; DQ167298; DQ167299; DQ167300; DQ167301;DQ167302; DQ167303; DQ167304; DQ167305; DQ167306; DQ167307; DQ174263;DQ174264; DQ174265; DQ174266; DQ174267; DQ174268; DQ179382; DQ179383;DQ179384; DQ179385; DQ179386; DQ179387; DQ179388; DQ179389; DQ179390;DQ179391; DQ179392; DQ179393; DQ179394; DQ179395; DQ179396; DQ179397;DQ179398; DQ179399; DQ179400; DQ179401; DQ179402; DQ179403; DQ179404;DQ179405; DQ179406; DQ179407; DQ179408; DQ179409; DQ179410; DQ179411;DQ179412; DQ179413; DQ179414; DQ179415; DQ179416; DQ179417; DQ179418;DQ179419; DQ179420; DQ179421; DQ179422; DQ179423; DQ179424; DQ179425;DQ179426; DQ179427; DQ179428; DQ179429; DQ179430; DQ179431; DQ179432;DQ179433; DQ179434; DQ179435; DQ179436; DQ179437; DQ179438; DQ179439;DQ179440; DQ179441; DQ179442; DQ179443; DQ179444; DQ179445; DQ179446;DQ179447; DQ179448; DQ179449; DQ179450; DQ179451; DQ179452; DQ179453;DQ179454; DQ179455; DQ179456; DQ179457; DQ179458; DQ179459; DQ179460;DQ179461; DQ179462; DQ179463; DQ179464; DQ179465; DQ179466; DQ179467;DQ179468; DQ179469; DQ179470; DQ179471; DQ179472; DQ179473; DQ179474;DQ179475; DQ179476; DQ179477; DQ179478; DQ179479; DQ179480; DQ179481;DQ179482; DQ179483; DQ179484; DQ179485; DQ179486; DQ179487; DQ179488;DQ179489; DQ179490; DQ179491; DQ179492; DQ179493; DQ179494; DQ179495;DQ179496; DQ179497; DQ179498; DQ179499; DQ179500; DQ179501; DQ179502;DQ179503; DQ179504; DQ179505; DQ179506; DQ179507; DQ179508; DQ179509;DQ179510; DQ179511; DQ179512; DQ179513; DQ179514; DQ179515; DQ179516;DQ179517; DQ179518; DQ179519; DQ179520; DQ179521; DQ179522; DQ179523;DQ179524; DQ179525; DQ179526; DQ179527; DQ222913; DQ227423; DQ227424;DQ227425; DQ227426; DQ227427; DQ227428; DQ227429; DQ227430; DQ227431;DQ241761; DQ241762; DQ241763; DQ249259; DQ249261; DQ249262; DQ256372;DQ256373; DQ256374; DQ256375; DQ265707; DQ265708; DQ265709; DQ265710;DQ265711; DQ265712; DQ265713; DQ265714; DQ265715; DQ265716; DQ265717;DQ265718; DQ335771; DQ336006; DQ336007; DQ336008; DQ336009; DQ336010;DQ336011; DQ336012; DQ336013; DQ336014; DQ336015; DQ336016; DQ336017;DQ415319; DQ415320; DQ415321; DQ415322; DQ415323; DQ415324; DQ415325;DQ415326; DQ447186; DQ469962; DQ469970; DQ469978; DQ469986; DQ469994;DQ470002; DQ487340; DQ487341; DQ508825; DQ508833; DQ508849; DQ508865;DQ508929; DQ534420; DQ534421; DQ534422; DQ534423; DQ534424; DQ534425;DQ534426; DQ534427; DQ534428; DQ534429; DQ632594; DQ632595; DQ632596;DQ632597; DQ865945; DQ865946; DQ865947; DQ865948; DQ865949; DQ865950;DQ865951; DQ865952; DQ865953; DQ865954; DQ865955; DQ865956; DQ865957;DQ865958; DQ865959; DQ865960; DQ865961; DQ865962; DQ865963; DQ865964;DQ865965; DQ865966; DQ865967; DQ865968; DQ865969; DQ865970; DQ865971;DQ865972; DQ865973; DQ865974; DQ883582; DQ883583; DQ883584; DQ883585;DQ883586; DQ883587; DQ883588; DQ883589; DQ883590; DQ883591; DQ883592;DQ883593; DQ883594; DQ883595; DQ883596; DQ883597; DQ883598; DQ883599;DQ883600; DQ883601; DQ883602; DQ883603; DQ883604; DQ883605; DQ883606;DQ883607; DQ883608; DQ883609; DQ883610; DQ883611; DQ883612; DQ883613;DQ883614; DQ883615; DQ883616; DQ883617; DQ883618; DQ883619; DQ883620;DQ883621; DQ883622; DQ883623; DQ883624; DQ883625; DQ883626; DQ883627;DQ883628; DQ923506; DQ923507; DQ973305; DQ975252; DQ975253; DQ975254;DQ975255; DQ975256; DQ975257; DQ975258; DQ975259; DQ975260; DQ975261;DQ975262; DQ975263; DQ975264; DQ975265; DQ975266; DQ975267; DQ981740;DQ981741; DQ981742; DQ983746; DQ983747; DQ983748; DQ983749; DQ983750;DQ983751; DQ983752; DQ983753; DQ983754; DQ983755; DQ983756; DQ983757;DQ983758; DQ983759; DQ983760; DQ983761; DQ983762; DQ983763; DQ983764;DQ983765; DQ983766; DQ983767; EF041487; EF117330; EF118172; EF118173;EF118174; EF151958; EF199897; EF199898; EF456782; EF456783; EF456784;EF456785; EF456786; EF456787; EF456788; EF456789; EF456790; EF456791;EF456792; EF456797; EF462544; EF462549; EF462550; EF462551; EF462552;EF462553; EF462554; EF462555; EF462557; EF462558; EF462559; EF462560;EF462561; EF462562; EF462566; EF462567; EF462568; EF462569; EF467799;EF467800; EF467827; EF473329; EF473330; EF473331; EF473332; EF473333;EF473334; EF473335; EF473336; EF473337; EF473338; EF473339; EF473340;EF473341; EF473342; EF473343; EF473344; EF473345; EF473346; EF473347;EF473348; EF473349; EF473350; EF473351; EF473352; EF473353; EF473354;EF473355; EF473356; EF473357; EF473358; EF473359; EF473360; EF473362;EF473363; EF473364; EF473365; EF473366; EF473367; EF473368; EF473369;EF473370; EF473371; EF473372; EF473373; EF473375; EF473376; EF473377;EF473378; EF473379; EF473380; EF473381; EF473382; EF473383; EF473384;EF473385; EF473386; EF473387; EF473388; EF473389; EF473390; EF473391;EF473392; EF473393; EF473394; EF473395; EF473396; EF473398; EF473399;EF473400; EF473401; EF473402; EF473403; EF473404; EF473405; EF473406;EF473408; EF473409; EF473410; EF473411; EF473412; EF473413; EF473414;EF473415; EF473416; EF473417; EF473418; EF473419; EF473420; EF473421;EF473422; EF473423; EF473424; EF473425; EF473426; EF473427; EF473428;EF473429; EF473430; EF473431; EF473432; EF473433; EF473434; EF473435;EF473436; EF473437; EF473438; EF473439; EF473440; EF473441; EF473442;EF473443; EF473444; EF473445; EF473446; EF473447; EF473449; EF473450;EF473451; EF473452; EF473453; EF473454; EF473455; EF473456; EF473457;EF473458; EF473459; EF473460; EF473461; EF473462; EF473463; EF473464;EF473465; EF473466; EF473467; EF473468; EF473469; EF473470; EF473471;EF473472; EF473473; EF473474; EF473475; EF473476; EF473477; EF473478;EF473479; EF473480; EF473481; EF473482; EF473483; EF473484; EF473485;EF473486; EF473487; EF473488; EF473489; EF473490; EF473491; EF473492;EF473493; EF473494; EF473495; EF473496; EF473497; EF473498; EF473499;EF473500; EF473504; EF473505; EF473506; EF473507; EF473508; EF473509;EF473510; EF473511; EF473512; EF473513; EF473514; EF473515; EF473516;EF473517; EF473518; EF473519; EF473520; EF473521; EF473522; EF473523;EF473524; EF473525; EF473526; EF473527; EF473528; EF473529; EF473530;EF473531; EF473532; EF473533; EF473534; EF473535; EF473536; EF473537;EF473538; EF473539; EF473540; EF473541; EF473542; EF473543; EF473544;EF473545; EF473546; EF473547; EF473548; EF473549; EF473550; EF473551;EF473552; EF473553; EF473555; EF473556; EF473557; EF473558; EF473559;EF473560; EF473561; EF473562; EF473563; EF473564; EF473565; EF473566;EF473567; EF473568; EF473569; EF473570; EF473571; EF473572; EF473573;EF473574; EF473575; EF473576; EF473577; EF473578; EF473579; EF473581;EF473582; EF473583; EF473584; EF473585; EF473586; EF473588; EF473589;EF473590; EF473591; EF473592; EF473593; EF473594; EF473595; EF473596;EF473597; EF473598; EF473599; EF473600; EF473601; EF473602; EF473603;EF473604; EF473605; EF473607; EF473608; EF473609; EF473611; EF473612;EF473613; EF473614; EF473615; EF473616; EF473617; EF473618; EF473619;EF473620; EF473621; EF473622; EF473623; EF473624; EF473625; EF473626;EF473627; EF473628; EF473629; EF473630; EF473632; EF473633; EF473634;EF473635; EF473636; EF473638; EF473639; EF473640; EF473641; EF473642;EF473643; EF473644; EF473645; EF473646; EF473647; EF473648; EF541428;EF541429; EF541430; EF541431; EF541432; EF541433; EF541434; EF541435;EF541436; EF541437; EF541438; EF541439; EF541440; EF541441; EF541442;EF541443; J02090; J02092; J02132; J02538; K03335; K03338; L18994;L18996; L18997; L18998; L19000; L9001; L19002; L19003; L19004; L19412;L19413; L19414; L19415; L19416; L20101; L20102; L20103; L20104; L20105;L20114; L20115; L20118; L20119; L27597; L31949; L32024; L39913; L39914;L39915; L39916; L39917; L39918; L75975; L75976; L75977; L75978; L75979;L75980; L75981; L75982; L75983; L75984; L75985; L75986; L75987; L75988;L75989; L75990; L75991; L76035; L76036; L76037; M16737; M16738; M16739;M16740; M16741; M16742; M16743; M19056; M19057; M21648; M24718; M24719;M24720; M24721; M24722; M24723; M24724; M24725; M24726; M24727; M24728;M25044; M25434; M29257; M54895; M55059; M57630; M57631; M57632; M57644;M65018; M73771; M73772; M73773; M73774; M73775; M73776; S64310; S77429;U07146; U08858; U08859; U08905; U26830; U48439; U48440; U48441; U48442;U48443; U48444; U48445; U48446; U48447; U49722; U58195; U65552; U65553;U65554; U65555; U65556; U65557; U65558; U65559; U65560; U77830; U77831;U77832; U77833; U77834; U77835; U77836; U77837; U77838; U77839; U77840;U97740; V01085; V01086; V01087; V01089; V01098; V01103; X05907; X68437;X73489; X73490; X73491; X75800; X85085; X85086; X85087; X85088; X85089;X85090; X95637; X95638; Y14053; Y14055; Y14056; Y14057; Y14058; Y14059;Y14060; Z46391; Z46392; Z46393; Z46394; Z46395; Z46396; Z46397; Z46398;Z46399; Z46400; Z46401; Z46402; Z46403; Z46404; Z46405; Z46406; Z46407;Z46408; Z46409; Z46410; Z46411; Z46412; Z46413; Z46414; Z46415; Z46416and Z46417.

Polynucleotides encoding influenza virus subtype H4 hemagglutininssuitable for inclusion as a cargo moiety in conjugates herein have thefollowing accession numbers and are available to the public via theNational Center for Biotechnology Information (NCBI) Entrez nucleotidesearch and retrieval system:

AB288842; AB289331; CY005952; CY005953; CY005954; CY005955; CY005956;CY005957; CY005958; CY005959; CY005961; CY005962; CY005963; CY005964;CY005965; CY005966; CY005967; CY005968; CY006017; CY006027; CY006030;CY011036; CY011056; CY012808; CY012816; CY013248; CY014562; CY014579;CY014630; CY014723; CY014751; CY014857; CY014922; CY014929; CY014937;CY015459; CY015467; CY016148; CY017701; CY017741; CY020725; CY020733;CY020749; CY020765; CY020773; CY020789; CY020797; CY020805; CY020981;CY021213; CY021221; CY021237; CY021325; CY021333; CY021349; D90302;DQ021848; DQ021849; DQ021850; DQ021851; DQ021852; DQ021853; DQ021854;DQ021855; DQ021856; DQ021857; DQ021858; DQ021859; DQ021860; DQ021861;DQ021862; DQ021863; DQ021864; DQ021865; DQ021866; DQ021867; DQ021868;DQ236166; DQ327834; DQ787806; EF041495; J02102; M25283; M25284; M25285;M25286; M25287; M25288; M25289; M25290; M25291; AB289333; AB292406;AB292408; AB292662; AB295609; AB295611; AF285883; AF285885; AF290436;AJ506780; AJ506782; AY180434; AY180435; AY180436; AY180437; AY180438;AY180439; AY180440; AY180441; AY180442; AY180443; AY596802; AY596803;AY596804; AY633124; AY633141; AY633156; AY633260; AY633268; AY633284;AY633348; AY633356; CY004847; CY004911; CY004925; CY004933; CY004939;CY005672; CY005679; CY005944; CY005945; CY005946; CY005947; CY005948;CY005950 and CY005951.

Polynucleotides encoding influenza virus subtype H5 hemagglutininssuitable for inclusion as a cargo moiety in conjugates herein have thefollowing accession numbers and are available to the public via theNational Center for Biotechnology Information (NCBI) Entrez nucleotidesearch and retrieval system:

BAD89305; BAD89315; BAD89325; BAD89335; BAD89345; BAE07201; BAE07155;BAE47131; BAE48315; BAE48316; BAE48317; BAE48318; BAE46949; BAE48684;BAE48685; BAE48686; BAE48687; BAE48688; BAE48689; BAE48690; BAE48691;BAE48692; BAE48693; BAE48694; BAE48695; BAE48696; BAE94699; BAF37962;BAE96567; BAE96961; BAF49662; BAF49663; BAF49664; BAF49665; BAF49666;BAF49667; BAF49668; BAF49669; BAF49670; BAF49671; BAF49672; BAF49673;BAF49674; BAF49675; BAF49676; BAF37387; BAF48359; AAC40508; AAC34263;AAC32078; AAC32088; AAC32098; AAC32099; AAC32100; AAC32101; AAC32102;AAC14418; AAD13566; AAD13567; AAD13568; AAD13569; AAD13570; AAD13571;AAD13572; AAD13573; AAD13574; AAD13575; AAF74329; AAF74330; AAF74331;AAD52043; AAF02300; AAF02301; AAF02302; AAF02303; AAF02304; AAF02305;AAF02306; AAF02307; AAF02308; AAF02309; AAF04719; AAF04720; AAD21153;AAD21154; AAD21155; AAD21156; AAD21157; AAD21158; AAD21159; AAD21160;AAD21161; AAD21162; AAD21163; AAD21164; AAD51927; AAD37782; AAF89536;AAF89537; AAF89538; AAF89539; AAF89540; AAF89541; AAF89542; AAF89543;AAF89544; AAF89545; AAF89546; AAG28424; AAG60347; AAG60348; AAG60349;AAG01195; AAG01205; AAG01215; AAG01225; AAF99718; AAG38534; AAK38298;AAK57506; AAL59142; AAL59143; AAL16033; AAL84323; AAL84324; AAM49555;AAM22457; AAM22458; AAO52859; AAO52860; AAO52861; AAO52862; AAO52863;AAO52864; AAO52865; AAO52866; AAO52867; AAO52868; AAO52869; AAO52870;AAO52871; AAO52872; AAO52873; AAO52874; AAO52875; AAO52876; AAO52877;AAO52878; AAO52879; AAO52880; AAO52881; AAO52882; CAC28131; CAF21870;CAF21874; CAG14996; CAG14997; CAG29661; CAI29278; CAI96162; CAI96163;CAI99404; CAJ32556; CAJ75440; CAJ75441; CAJ75442; CAJ75443; CAJ75444;CAJ75445; CAJ75446; CAJ75447; CAJ75448; CAJ77761; CAJ84721; CAK18565;CAK18566; CAK18567; CAK18570; CAK18571; CAK18577; CAK18596; CAL37103;CAL51387; CAL51388; CAL51389; CAL51390; CAL51391; CAL51392; CAL51393;CAL51394; CAL51395; CAL51396; CAL48277; CAL48276; CAL48275; CAL48274;CAL48273; CAL48272; CAL48271; CAL48270; CAL48269; CAL48268; CAL48267;CAL48279; CAL48278; CAL48266; CAL48265; CAL48280; CAL59784; CAL59783;CAL59782; CAL59781; CAL59780; CAL59779; CAL59786; CAL59785; CAM33521;AAL31380; AAL31381; AAL31382; AAL31383; AAL31384; AAL31385; AAL31386;AAL31387; AAL31388; AAL75839; AAL75843; AAL75847; AAO46797; AAO46798;AAO46799; AAO46800; AAO46801; AAO46802; AAO46803; AAO46804; AAO46805;AAP71989; AAP71990; AAP71991; AAP71992; AAP71993; AAP71994; AAP71995;AAP71996; AAP71997; AAP71998; AAP71999; AAP72000; AAP72001; AAP72002;AAP72003; AAP72004; AAP72005; AAP72006; AAP72007; AAP72008; AAP72009;AAP72010; AAP72011; AAR88808; AAR88809; AAR88810; AAR88811; AAR88812;AAR88813; AAR88814; AAR88815; AAR88816; AAR88817; AAR88818; AAR88819;AAR88820; AAR88821; AAR88822; AAR88823; AAR88824; AAR88825; AAR88826;AAR88827; AAR88828; AAR88829; AAR88830; AAR88831; AAR88832; AAR88833;AAR88834; AAR88835; AAR88836; AAR88837; AAR88838; AAR88839; AAR88840;AAR88841; AAS07023; AAR99628; AAR98819; AAT07996; AAS50166; AAS50167;AAS57873; AAS57874; AAS57875; AAS57876; AAS45134; AAS84275; AAS84276;AAS84247; AAS84248; AAS84249; AAS84250; AAS84251; AAS84252; AAS84253;AAS84254; AAS84255; AAS84256; AAS84257; AAS84258; AAS84259; AAS84260;AAS84261; AAS84262; AAS84263; AAS84264; AAS84265; AAS84266; AAS84267;AAS84268; AAS84269; AAS84270; AAS84271; AAS84272; AAS84273; AAS84274;AAS65615; AAS65618; AAS87596; AAS87577; AAS87580; AAT39065; AAT39066;AAT39067; AAT39068; AAT39073; AAT39074; AAT39075; AAT39076; AAT39077;AAT39078; AAT39079; AAT39080; AAS79356; AAS79359; AAS89004; AAT12022;AAT12023; AAT12024; AAT12025; AAT12026; AAT12027; AAT12028; AAT12029;AAT12030; AAT12031; AAT12032; AAT12033; AAT12034; AAT12035; AAT12036;AAT12037; AAT12038; AAT12039; AAT12040; AAT12041; AAT12042; AAS89267;AAS89268; AAS89269; AAS89270; AAS89271; AAS89272; AAS89273; AAT37563;AAT90337; AAV34704; AAV32636; AAT65209; AAT70210; AAT70218; AAT72505;AAV65826; AAT73260; AAT73261; AAT73262; AAT73263; AAT73264; AAT73265;AAT73266; AAT73267; AAT73268; AAT73269; AAT73270; AAT73271; AAT73272;AAT73273; AAT73274; AAT73275; AAT73276; AAT73277; AAT73278; AAT73279;AAT73280; AAT73281; AAT73282; AAT73283; AAT73284; AAT73285; AAT73286;AAT73287; AAT73288; AAT73289; AAT73290; AAT73291; AAT73292; AAT73293;AAT73294; AAT73295; AAT73296; AAT73297; AAT73298; AAT73299; AAT73300;AAT73301; AAT73302; AAT73303; AAT73304; AAT73305; AAT73306; AAT73307;AAT73308; AAT73309; AAT73310; AAT73311; AAT73312; AAT73313; AAT76166;AAV97601; AAV97602; AAV97603; AAV97604; AAT84153; AAT90832; AAV91220;AAV73972; AAV73975; AAV73980; AAW59548; AAW59550; AAW59552; AAW59554;AAW59556; AAW59558; AAW59559; AAU08349; AAU08351; AAW59390; AAW59398;AAW59408; AAW19638; AAW19640; AAW19642; AAW19644; AAW19646; AAV30828;AAV30836; AAV48546; AAV41002; AAV48778; AAV48780; AAV74400; AAW80717;AAW80718; AAW80719; AAV91149; AAV97886; AAW30657; AAX47288; AAW72226;AAX59694; AAW66002; AAX53504; AAX53505; AAX53506; AAX53507; AAX53508;AAX53509; AAX53510; AAX83395; AAX83396; AAX83397; AAX83398; AAY57183;AAY57184; AAY57185; AAY57186; AAY57187; AAY57188; AAY57189; AAY57190;AAY57191; AAY57192; AAY57193; AAY57194; AAY57195; AAY57196; AAY57197;AAY57198; ABB20262; ABB87042; ABB87281; ABB87292; ABB87711; ABB88278;ABB88348; ABB88379; ABG88245; ABI36041; ABI36012; ABI36023; ABI36034;ABI36040; ABI36042; ABI36043; ABI36044; ABI36045; ABI36046; ABI36047;ABI36048; ABI36049; ABI36050; ABI36051; ABI36052; ABI36053; ABI36054;ABI36055; ABI36056; ABI36057; ABI36144; ABI36155; ABI36166; ABI36177;ABI36187; ABI36198; ABI36275; ABI36286; ABI36295; ABI36307; ABI36318;ABI36329; ABI36340; ABI36351; ABI36362; ABI36373; ABI36384; ABI36395;ABI36406; ABI36423; ABI36428; ABI36439; ABI36450; ABI36469; ABI36480;ABI49396; ABI49407; ABI49415; ABI84424; ABI84465; ABI84495; ABI84497;ABI84598; ABI84603; ABI84608; ABI84784; ABI84816; ABI84970; ABI85095;ABI85106; ABI85117; ABI85155; ABI95316; ABI95327; ABI95338; ABI95349;ABJ16565; ABJ16796; ABJ16807; ABJ16818; ABJ16928; ABJ16917; ABJ16829;ABJ16939; ABJ51728; ABJ51717; ABJ51706; ABJ51695; ABJ51739; ABJ51684;ABJ51673; ABJ16950; ABJ16840; ABJ16851; ABJ16862; ABJ16873; ABJ16884;ABJ16895; ABJ16906; ABJ53526; ABJ53537; ABJ53548; ABJ53594; ABJ53583;ABJ53559; ABK40087; ABK40492; ABK80003; ABL07008; ABL07019; ABL07030;ABL31744; ABL31755; ABL31766; ABL31780; ABM22048; ABM90434; ABM90445;ABM90456; ABM90467; ABM90478; ABM90489; ABM90500; ABM90511; ABM90522;ABM90533; ABM90544; ABO37977; ABO38263; ABO44200; ABO44211; ABO44222;ABO44233; ABO44244; ABO44255; ABO44266; ABO44277; ABO44288; ABO44299;ABO44310; ABO52720; ABO52731; ABO52742; ABO52753; ABO77034; ABO77045;AAY21163; AAY25499; AAY46328; AAY46329; AAY46330; AAY46331; AAY46332;AAY46333; AAY46334; AAY46335; AAY46336; AAY46337; AAY46338; AAY46339;AAY46340; AAY46341; AAY46342; AAY46343; AAY46344; AAY46345; AAY46346;AAY46347; AAY46348; AAY46349; AAY46350; AAY46351; AAY46352; AAY46353;AAY46354; AAY46355; AAY46356; AAY46357; AAY46358; AAY46359; AAY46360;AAY46361; AAY46362; AAY46363; AAY46364; AAY46365; AAY46366; AAY56367;AAY68363; AAY78953; AAZ29946; AAZ29947; AAZ29948; AAZ29949; AAZ29950;AAZ29951; AAZ29952; AAZ29953; AAZ29954; AAZ29955; AAZ29956; AAZ29957;AAZ29958; AAZ29959; AAZ29960; AAZ29961; AAZ29962; AAZ29963; AAZ29964;AAZ29965; AAZ29966; AAZ29967; AAZ29968; AAZ29969; AAZ29970; AAZ29971;AAZ29972; AAZ29973; AAZ29974; AAZ29975; AAZ29976; AAZ29977; AAZ29978;AAZ29979; AAZ29980; AAZ29981; AAZ76389; ABE68921; ABE68922; AAZ16275;ABE68923; ABE68924; ABE68925; ABE68926; AAZ16276; AAZ16277; ABE68927;AAZ16278; AAZ16279; ABE68928; ABE68929; AAZ16280; ABE68930; ABE68931;ABE68932; AAZ16281; AAZ16282; AAZ72734; AAZ72735; AAZ72736; AAZ72737;AAZ72738; AAZ72739; AAZ17522; AAZ17523; AAZ17524; AAZ23154; AAZ80486;AAZ78315; ABA29447; AAZ82496; AAZ82497; ABA70758; ABB00917; ABB00918;ABB00919; ABB00920; ABA39516; ABA39517; ABA39518; ABA39519; ABA39520;ABA87102; ABA87103; ABA54915; ABA55714; ABA55715; ABA55716; ABA55717;ABB00582; ABB43058; ABB43059; ABB22773; ABB22774; ABB22775; ABB43119;ABB43127; ABB83598; ABB58817; ABB58818; ABB58819; ABB58820; ABB58821;ABB80546; ABB86287; ABC47656; ABC59833; ABC66517; ABC66518; ABC66519;ABC66520; ABC66521; ABC66522; ABC66523; ABC66524; ABC66525; ABC66526;ABC66527; ABC66528; ABC66529; ABC66530; ABC66531; ABC66532; ABC66533;ABC66534; ABC66535; ABC66536; ABC66537; ABC66538; ABC66539; ABC66540;ABC66541; ABC66542; ABC66543; ABC66544; ABC66545; ABC66546; ABC66547;ABC66548; ABC66549; ABC66550; ABC66551; ABC66552; ABC66553; ABC66554;ABC66555; ABC66556; ABC66557; ABC66558; ABC66559; ABC66560; ABC66561;ABC66562; ABC66563; ABC66564; ABC66565; ABC66566; ABC66567; ABC66568;ABC66569; ABC66570; ABC66571; ABC66572; ABC66573; ABC66574; ABC66575;ABC66576; ABC66577; ABC66578; ABC66579; ABC66580; ABC66581; ABC66582;ABC48787; ABC69216; ABC69224; ABC69232; ABC70167; ABC69148; ABC69149;ABC69150; ABC70712; ABC72082; ABC87315; ABC72655; ABD32123; ABD32128;ABC88573; ABC88583; ABD14806; ABD14807; ABD14808; ABD14809; ABD14810;ABD28180; ABD28181; ABD28182; ABD16284; ABD46889; ABD49489; ABD60336;ABD60345; ABD46740; ABD73284; ABD52284; ABD65415; ABD66291; ABD66292;ABD66293; ABD73804; ABD85144; ABD83818; ABD92945; ABD92953; ABD85374;ABD95991; ABE26829; ABE01046; ABE97547; ABE97548; ABE97549; ABE97550;ABE97551; ABE97552; ABE97553; ABE97554; ABE97555; ABE97556; ABE97557;ABE97558; ABE97559; ABE97560; ABE97561; ABE97562; ABE97563; ABE97564;ABE97565; ABE97566; ABE97567; ABE97568; ABE97569; ABE97570; ABE97571;ABE97572; ABE97573; ABE97574; ABE97575; ABE97576; ABE97577; ABE97578;ABE97579; ABE97580; ABE97581; ABE97582; ABE97583; ABE97584; ABE97585;ABE97586; ABE97587; ABE97588; ABE97589; ABE97590; ABE97591; ABE97592;ABE97593; ABE97594; ABE97595; ABE97596; ABE97597; ABE97598; ABE97599;ABE97600; ABE97601; ABE97602; ABE97603; ABE97604; ABE97605; ABE97606;ABE97607; ABE97608; ABE97609; ABE97610; ABE97611; ABE97612; ABE97613;ABE97614; ABE97615; ABE97616; ABE97617; ABE97618; ABE97619; ABE97620;ABE97621; ABE97622; ABE97623; ABE97624; ABE97625; ABE97626; ABE97627;ABE97628; ABE97629; ABE97630; ABE97631; ABE97632; ABE97633; ABE97634;ABF56528; ABF58847; ABF56648; ABG23657; ABF61761; ABG20463; ABG20464;ABG20465; ABG20466; ABG20467; ABG38185; ABG38189; ABF72802; ABF93440;ABF93441; ABG49439; ABF84066; ABG45944; ABG75543; ABG20468; ABG20472;ABG20476; ABG20478; ABG35546; ABG65732; ABI16504; ABG65733; ABG67711;ABG67712; ABG67713; ABG67714; ABG57086; ABG57087; ABG57094; ABG57095;ABG78549; ABG78567; ABI34140; ABI34142; ABG67978; ABG75831; ABG75616;ABI23979; ABG81037; ABG81038; ABG81039; ABG81040; ABG81041; ABI18096;ABH85395; ABH09484; ABH09485; ABH09486; ABH09487; ABH09488; ABH09489;ABH09490; ABJ98523; ABJ98525; ABJ98527; ABJ98529; ABJ98531; ABI34124;ABK34764; ABJ88847; ABJ96647; ABJ96648; ABJ96649; ABJ96650; ABJ96651;ABJ96652; ABJ96653; ABJ96654; ABJ96655; ABJ96656; ABJ96657; ABJ96658;ABJ96659; ABJ96660; ABJ96661; ABJ96662; ABJ96663; ABJ96664; ABJ96665;ABJ96666; ABJ96667; ABJ96668; ABJ96669; ABJ96670; ABJ96671; ABJ96672;ABJ96673; ABJ96674; ABJ96675; ABJ96676; ABJ96677; ABJ96678; ABJ96679;ABJ96680; ABJ96681; ABJ96682; ABJ96683; ABJ96684; ABJ96685; ABJ96686;ABJ96687; ABJ96688; ABJ96689; ABJ96690; ABJ96691; ABJ96692; ABJ96693;ABJ96694; ABJ96695; ABJ96696; ABJ96697; ABJ96698; ABJ96699; ABJ96700;ABJ96701; ABJ96702; ABJ96703; ABJ96704; ABJ96705; ABJ96706; ABJ96707;ABJ96708; ABJ96709; ABJ96710; ABJ96711; ABJ96712; ABJ96713; ABJ96714;ABJ96715; ABJ96716; ABJ96717; ABJ96718; ABJ96719; ABJ96720; ABJ96721;ABJ96722; ABJ96723; ABJ96724; ABJ96725; ABJ96726; ABJ96727; ABJ96728;ABJ96729; ABJ96730; ABJ96731; ABJ96732; ABJ96733; ABJ96734; ABJ96735;ABJ96736; ABJ96737; ABJ96738; ABJ96739; ABJ96740; ABJ96741; ABJ96742;ABJ96743; ABJ96744; ABJ96745; ABJ96746; ABJ96747; ABJ96748; ABJ96749;ABJ96750; ABJ96751; ABJ96752; ABJ96753; ABJ96754; ABJ96755; ABJ96756;ABJ96757; ABJ96758; ABJ96759; ABJ96760; ABJ96761; ABJ96762; ABJ96763;ABJ96764; ABJ96765; ABJ96766; ABJ96767; ABJ96768; ABJ96769; ABJ96770;ABJ96771; ABJ96772; ABJ96773; ABJ96774; ABJ96775; ABJ96776; ABJ96777;ABJ96778; ABJ96779; ABJ96780; ABJ96781; ABJ96782; ABJ96783; ABJ96784;ABJ96785; ABJ96786; ABJ96787; ABJ96788; ABJ96789; ABJ96790; ABJ96791;ABJ96792; ABJ96793; ABJ96794; ABJ96795; ABJ96796; ABJ96797; ABJ96798;ABJ96799; ABJ96800; ABJ96801; ABJ96802; ABJ96803; ABJ96804; ABJ96805;ABJ96806; ABJ96807; ABJ96808; ABJ96809; ABJ96810; ABJ96811; ABJ96812;ABJ96813; ABJ96814; ABJ96815; ABJ96816; ABJ96817; ABJ96818; ABJ96819;ABJ96820; ABJ96821; ABJ96822; ABJ96823; ABJ96824; ABJ96825; ABJ96826;ABJ96827; ABJ96828; ABJ96829; ABJ96830; ABJ96831; ABJ96832; ABJ96833;ABJ96834; ABJ96835; ABJ96836; ABJ96837; ABJ96838; ABJ96839; ABJ96840;ABJ96841; ABJ96842; ABJ96843; ABJ96844; ABJ96845; ABJ96846; ABJ96847;ABJ96848; ABJ96849; ABJ96850; ABJ96851; ABJ96852; ABJ96853; ABJ96854;ABJ96855; ABJ96856; ABJ96857; ABJ96858; ABJ96859; ABJ96860; ABJ96861;ABJ96862; ABJ96863; ABJ96864; ABJ96865; ABJ96866; ABJ96867; ABJ96868;ABJ96869; ABJ96870; ABJ96871; ABJ96872; ABJ96873; ABJ96874; ABJ96875;ABJ96876; ABJ96877; ABJ96878; ABJ96879; ABJ96880; ABJ96881; ABJ96882;ABJ96883; ABJ96884; ABJ96885; ABJ96886; ABJ96887; ABJ96888; ABJ96889;ABJ96890; ABJ96891; ABJ96892; ABJ96893; ABJ96894; ABJ96895; ABJ96896;ABJ96897; ABJ96898; ABJ96899; ABJ96900; ABJ96901; ABJ96902; ABJ96903;ABJ96904; ABJ96905; ABJ96906; ABJ96907; ABJ96908; ABJ96909; ABJ96910;ABJ96911; ABJ96912; ABJ96913; ABJ96914; ABJ96915; ABJ96916; ABJ96917;ABJ96918; ABJ96919; ABJ96920; ABJ96921; ABJ96922; ABJ96923; ABJ96924;ABJ96925; ABJ96926; ABJ96927; ABJ96928; ABJ96929; ABJ96930; ABJ96931;ABJ96932; ABJ96933; ABJ96934; ABJ96935; ABJ96936; ABJ96937; ABJ96938;ABJ96939; ABJ96940; ABJ96941; ABJ96942; ABJ96943; ABJ96944; ABJ96945;ABJ96946; ABJ96947; ABJ96948; ABJ96949; ABJ96950; ABJ96951; ABJ96952;ABJ96953; ABJ96954; ABJ96955; ABJ96956; ABJ96957; ABJ96958; ABJ96959;ABJ96960; ABJ96961; ABJ96962; ABJ96963; ABJ96964; ABJ96965; ABJ96966;ABJ96967; ABJ96968; ABJ96969; ABJ96970; ABJ96971; ABJ96972; ABJ96973;ABJ96974; ABJ96975; ABJ96976; ABJ96977; ABJ96978; ABJ96979; ABJ96980;ABJ96981; ABJ96982; ABJ96983; ABJ96984; ABJ96985; ABJ96986; ABJ96987;ABJ96988; ABJ96989; ABJ96990; ABJ96991; ABJ96992; ABJ96993; ABJ96994;ABJ96995; ABJ96996; ABJ96997; ABJ96998; ABJ96999; ABJ97000; ABJ97001;ABJ97002; ABJ97003; ABJ97004; ABJ97005; ABJ97006; ABJ97007; ABJ97008;ABJ97009; ABJ97010; ABJ9711; ABJ97012; ABJ97013; ABJ97014; ABJ97015;ABJ97016; ABJ97017; ABJ97018; ABJ97019; ABJ97020; ABJ97021; ABJ97022;ABJ97023; ABJ97024; ABJ97025; ABJ97026; ABJ97027; ABJ97028; ABJ97029;ABJ97030; ABJ97031; ABJ97032; ABJ97033; ABJ97034; ABJ97035; ABJ97036;ABJ97037; ABJ97038; ABJ97039; ABJ97040; ABJ97041; ABJ97042; ABJ97043;ABJ97044; ABJ97045; ABJ97046; ABJ97047; ABJ97048; ABJ97049; ABJ97050;ABK000133; ABI94741; ABI94747; ABI94754; ABI94764; ABI96729; ABI96730;ABI96741; ABJ09476; ABI96767; ABJ09545; ABI96701; ABJ16473; ABJ15720;ABI98911; ABJ09528; ABI98919; ABI97335; ABJ52562; ABJ80592; ABK00083;ABK00087; ABK00096; ABI98929; ABK000132; ABI97303; ABJ09511; ABJ09498;ABJ09466; ABJ09518; ABJ09488; ABK000104; ABI98938; ABK13783; ABK13784;ABK13782; ABJ53148; ABK32775; ABK32776; ABK32777; ABK32778; ABK32779;ABK32780; ABK32781; ABK32782; ABK34511; ABK34512; ABK34513; ABJ90343;ABK79301; ABK79302; ABK79303; ABK79304; ABL10088; ABL74499; ABL74500;ABL75919; ABL63754; ABL63755; ABL63756; ABL63757; ABL63758; ABL63759;ABL63760; ABL63761; ABL63762; ABL63763; ABL63764; ABL63765; ABL63766;ABL63767; ABL63768; ABL63769; ABL63770; ABL63771; ABL63772; ABM54179;ABM54180; ABO76638; ABO76639; ABO76640; ABO76641; ABO76642; ABO76643;ABO76644; ABM92273; ABN54791; ABN54792; ABO14789; ABO14790; ABO30505;ABN70706; ABN70707; ABN70708; ABN70709; ABN70710; ABN70711; ABO13912;ABO13920; ABO38179; ABO20946; ABO10162; ABO10163; ABO10181; ABO10183;ABO10184; ABO10185; ABO10186; ABO10187; ABO20962; ABO64687; ABO64688;ABO64689; ABO64690; ABO64691; ABO64692; ABO64693; ABO64694; ABO64695;ABO64696; ABO64697; ABO30353; ABO30354; ABO30355; ABO30359; ABO30360;ABO30361; ABO30346; ABO30347; ABO31434; AAA43199; AAA43094; AAL34297;AAL34298; AAL34299; AAA43159; AAA43160; AAA43082; AAA43083; AAA43205;AAB29507; AAB82064; AAA74909; AAA74910; AAC54378; AAC54390; AAC54391;AAC54392; AAC54393; AAB49654; AAB49655; AAB19072; AAB19073; AAB19074;AAB19075; AAB19076; AAB19077; AAB19078; AAB19079; AAB19080; AAB19081;AAB19082; AAB19083; AAB19084; AAB19085; AAB19086; AAB19087; AAB19088;AAB19089; AAC58999; AAB39639; AAC58990; AAC58991; AAC58992; AAC58993;AAC58994; AAC58995; AAC58996; AAC58997; AAC58998; CAA30680 and CAA30719.

Polynucleotides encoding influenza virus subtype H6 hemagglutininssuitable for inclusion as a cargo moiety in conjugates herein have thefollowing accession numbers and are available to the public via theNational Center for Biotechnology Information (NCBI) Entrez nucleotidesearch and retrieval system:

AB278600; AB286875; AJ410532; AJ410533; AJ410534; AJ410535; AJ410536;AJ410537; AJ410538; AJ410539; AJ410540; AJ410541; AJ410542; AJ410543;AJ410544; AJ410545; AJ410546; AJ410547; AJ427308; AJ507203; AJ507204;AJ507205; AJ507206; AJ507207; AJ507208; AJ507209; AJ697867; AJ697868;AJ697869; AJ697870; AJ697871; AY633188; AY633204; AY633220; AY633236;AY633300; AY633308; AY633316; AY633324; AY633332; AY633380; AY684892;AY703832; AY773907; AY862613; AY968676; CY004034; CY004035; CY004036;CY004037; CY004038; CY004039; CY004043; CY004054; CY004066; CY004072;CY004076; CY004080; CY004086; CY004094; CY004114; CY004129; CY004137;CY004142; CY004146; CY004154; CY004162; CY004170; CY004178; CY004186;CY004194; CY004202; CY004210; CY004218; CY004226; CY004234; CY004242;CY004250; CY004258; CY004266; CY004274; CY004282; CY004515; CY004523;CY005106; CY005597; CY005605; CY005691; CY005881; CY01112; CY012832;CY013255; CY013863; CY014561; CY014607; CY014616; CY014623; CY014656;CY014764; CY014880; CY014888; CY014909; CY014945; CY014953; CY015127;CY015451; CY015476; CY015484; CY016124; CY016132; CY016156; CY016164;CY016172; CY016619; CY017789; CY018007; CY018893; CY018909; CY018917;CY020781; CY020813; CY020821; CY020829; CY020837; CY020845; CY020853;CY020869; CY020957; CY020973; CY020989; CY021197; CY021205; CY021477;CY021677; D90303; DQ021649; DQ021650; DQ021651; DQ021652; DQ021653;DQ021654; DQ021655; DQ021656; DQ021657; DQ021658; DQ021659; DQ021660;DQ021661; DQ021662; DQ021663; DQ021664; DQ021665; DQ021666; DQ021667;DQ021668; DQ021669; DQ021670; DQ021671; DQ021672; DQ021673; DQ021675;DQ021676; DQ021677; DQ021678; DQ021679; DQ021680; DQ021681; DQ021682;DQ021683; DQ021684; DQ285546; DQ376618; DQ376619; DQ376620; DQ376621;DQ376622; DQ376623; DQ376624; DQ376625; DQ376626; DQ376627; DQ376628;DQ376629; DQ376630; DQ376631; DQ376632; DQ376633; DQ376634; DQ376635;DQ376636; DQ376637; DQ376638; DQ376639; DQ376640; DQ376641; DQ376642;DQ376643; DQ376644; DQ376645; DQ376646; DQ376647; DQ376648; DQ376649;DQ376650; DQ376651; DQ376652; DQ376653; DQ408509; DQ408517; DQ408524;DQ822190; DQ822198; J02158; AB294213; AB294215; AB294219; AB295615;AB296072; AB298279; AF100181; AF250479; AF310983; AF310984; AF310985;AF457663; AF457664; AF457665; AF457666; AF457667; AF457668; AF457669;AF457670; AF457679; AF457688; AF457696; AF457704; AF457713; AF457715;AF474029; AF474030; AF474031; AF474032; AF474033; AF474034; AF474035;AF474036; AF474037; AF474038; AJ410519; AJ410520; AJ410521; AJ410522;AJ410523; AJ410524; AJ410525; AJ410526; AJ410527; AJ410528; AJ410529;AJ410530 and AJ410531.

Polynucleotides encoding influenza virus subtype H7 hemagglutininssuitable for inclusion as a cargo moiety in conjugates herein have thefollowing accession numbers and are available to the public via theNational Center for Biotechnology Information (NCBI) Entrez nucleotidesearch and retrieval system:

AB262459; AF072385; AF202256; AF322021; AF322022; AF322023; AF322024;AF322025; AF322026; AF364133; AF364134; AF364135; AF364136; AF364137;AF364138; AF364139; AF364140; AF364141; AF364142; AF364143; AF364144;AF364145; AF364146; AF364147; AF364148; AF364149; AF364150; AF364151;AF364152; AF364153; AF364154; AF364155; AF364156; AF364157; AF364158;AF364159; AF364160; AF364161; AF364162; AF364163; AF364164; AF364165;AF364166; AF364167; AF364168; AF364169; AF364170; AF364171; AF364172;AF497551; AF497552; AF497553; AF497554; AF497555; AF497556; AF497557;AF497558; AF497559; AJ489520; AJ491720; AJ493212; AJ493213; AJ493214;AJ493215; AJ493216; AJ493217; AJ493466; AJ493467; AJ493468; AJ493469;AJ493470; AJ493471; AJ493472; AJ580353; AJ584647; AJ620350; AJ627491;AJ627493; AJ697872; AJ697873; AJ704797; AJ704798; AJ704799; AJ704810;AJ704811; AJ704812; AJ704813; AM087214; AM087223; AY240877; AY240878;AY240879; AY240880; AY240881; AY240882; AY240883; AY240884; AY240885;AY240886; AY240887; AY240888; AY240889; AY240890; AY240891; AY240892;AY240893; AY240894; AY240895; AY240896; AY240897; AY240898; AY240899;AY240900; AY240901; AY240902; AY240903; AY240904; AY240905; AY240906;AY240907; AY240908; AY240909; AY240910; AY240911; AY240912; AY240913;AY240914; AY240915; AY240916; AY240917; AY240918; AY240919; AY240920;AY240921; AY240922; AY240923; AY240924; AY240925; AY303630; AY303631;AY303632; AY303633; AY303634; AY303635; AY338455; AY338456; AY338457;AY338458; AY338459; AY338460; AY338461; AY338462; AY383756; AY559235;AY586408; AY586409; AY586410; AY586411; AY596307; AY611524; AY644402;AY646078; AY648287; AY650270; AY672090; AY724257; AY724684; AY725855;AY730057; AY731820; AY734541; AY736323; AY831668; AY831669; AY831670;AY943924; AY999977; AY999978; AY999979; AY999980; AY999981; AY999982;AY999983; AY999984; AY999985; AY999986; AY999987; AY999988; AY999989;AY999990; AY999991; CY005928; CY005973; CY005974; CY005975; CY005976;CY005978; CY005980; CY005981; CY005983; CY006029; CY006037; CY014587;CY014612; CY014718; CY014721; CY014778; CY014786; CY014896; CY014992;CY015006; CY015014; CY015027; CY015033; CY015065; CY016188; CY018901;CY020581; CY020589; CY020597; CY020605; CY020613; CY020685; CY020885;CY021357; CY021365; CY021405; CY021413; CY021421; CY021485; CY021493;CY021501; CY021533; CY021541; CY021549; CY021557; CY021621; CY021637;DQ003216; DQ017504; DQ017513; DQ525411; DQ838510; DQ838511; DQ838512;DQ838513; DQ838514; DQ838515; DQ870888; DQ870894; DQ873807; DQ907527;DQ907528; DQ991304; DQ991312; DQ991320; DQ991328; DQ991336; DQ991343;EF467825; EF467826; J02164; K00429; L37794; L43913; L43914; L43915;M17735; M17736; M24457; M24458; M31689; M58657; U20458; U20459; U20461;U20462; U20463; U20464; U20465; U20466; U20467; U20468; U20469; U20470;U20471; X61627; X62552; X62553; X62554; X62555; X62556; X62557; X62558;X62559; X62560; Z12617; Z47199; AB262468; AB262469; AB262470; AB262471;AB262472; AB262473; AB268557; AB269692; AB269693; AB269694; AB269695;AB269696; AB269872; AB270592; AB270593; AB297923; AB297925; AB298277;AF028020; AF028021; AF071775; AF071776; AF072383; AF072384; AF072386;AF072387; AF072388; AF072389; AF072390; AF072391; AF072392; AF072393;AF072394; AF072395; AF072396; AF072397; AF072398; AF072399; AF072400;AF072401; AF072402; AF149295; AF202226; AF202227; AF202228; AF202229;AF202230; AF202231; AF202232; AF202233; AF202234; AF202235; AF202236;AF202237; AF202238; AF202239; AF202240; AF202241; AF202242; AF202243;AF202244; AF202245; AF202246; AF202247; AF202248; AF202249; AF202250;AF202251; AF202252; AF202253; AF202254 and AF202255.

Polynucleotides encoding influenza virus subtype H8 hemagglutininssuitable for inclusion as a cargo moiety in conjugates herein have thefollowing accession numbers and are available to the public via theNational Center for Biotechnology Information (NCBI) Entrez nucleotidesearch and retrieval system:

AB289343; AF310987; AF310988; AF310989; CY005970; CY005971; CY005972;CY014583; CY014659; CY015173; CY017749; D90304; EF061122 and J02089.

Polynucleotides encoding influenza virus subtype H9 hemagglutininssuitable for inclusion as a cargo moiety in conjugates herein have thefollowing accession numbers and are available to the public via theNational Center for Biotechnology Information (NCBI) Entrez nucleotidesearch and retrieval system:

J02166; AF156385; AF156386; AF156387; AF156388; AF156389; AF156390;AF156373; AF156374; AF156375; AF156376; AF156377; AF156378; AF156379;AF156380; AF156381; AF156382; AF156383; AF156384; AF186266; AF186267;AF186268; AF186269; AF203008; AF203009; AF203010; AF203011; AF203012;AF203013; AF203014; AF203015; AF222606; AF222607; AF222608; AF222609;AF222610; AF222611; AF222612; AF222613; AF218086; AF218087; AF218088;AF218089; AF218090; AF218091; AF218092; AF218093; AF218094; AF218095;AF218096; AF218097; AF218098; AF218099; AF218100; AF218101; AF218102;AF218103; AF218104; AF218105; AF218106; AF218107; AF218108; AF218109;AF218110; AF218111; AF218112; AF218113; AF218114; AF218115; AF218116;AF218117; AF218118; AF218119; AF218120; AF384557; AY036880; AF222810;AF222811; AF400776; AF400777; AY043014; AY043015; AY043017; AY043018;AY043019; AF461509; AF461510; AF461511; AF461512; AF461513; AF461514;AF461515; AF461516; AF461517; AF461518; AF461519; AF461520; AF461521;AF461522; AF461523; AF461524; AF461525; AF461526; AF461527; AF461528;AF461529; AF461530; AF461531; AF461532; AY083840; AY083841; AF536689;AF536690; AF536691; AF536692; AF536693; AF536694; AF536695; AF536696;AF536697; AF536698; AY180444; AY180445; AY180446; AY180447; AY180448;AY180449; AY180450; AY180451; AY180452; AY180453; AY180454; AY180455;AY180456; AY180457; AY180458; AY180459; AY206671; AY206672; AY206673;AY206674; AY206675; AY206676; AY206677; AY206678; AY206679; AY206680;AY198313; AY198314; AY198315; AY198316; AY198317; AY198318; AY198319;AY198320; AY198321; AY281745; AY264870; AY264871; AY264872; AY264875;AY264876; AY294658; AF523372; AF523373; AF523374; AF523375; AF523376;AF523377; AF523378; AF523379; AF523380; AF523381; AF523382; AF523383;AF523384; AF523385; AF523386; AF523387; AF523388; AF523389; AF523390;AY336597; AF508554; AF508555; AF508556; AF508557; AF508558; AF508559;AF508560; AF508561; AF508562; AF508563; AF508564; AF508565; AF508566;AF508567; AF508568; AF508569; AF508570; AF508571; AF508572; AF508573;AF508574; AY345925; AY345926; AY345927; AY345928; AY345929; AY345930;AY345931; AY345932; AY345933; AY345934; AY345935; AY345936; AY345937;AY345938; AY345939; AY345940; AY364228; AY330332; AY330333; AY330334;AY330335; AY330336; AY435039; AY435040; AY513715; AY548499; AY548500;AY548501; AY548502; AY548503; AY548504; AY548505; AY548506; AY548507;AY548508; AY548509; AY548510; AY548511; AY548512; AY548513; AY548514;AY548515; AY603067; AY549889; AY623810; AY633116; AY633164; AY633276;AY633292; AY652980; AY594194; AY594195; AY594196; AY664660; AY664661;AY664662; AY664663; AY664664; AY664665; AY664666; AY664667; AY664668;AY664669; AY664670; AY664671; AY664672; AY664673; AY664674; AY664675;AY664676; AY664677; AY664678; AY743216; AY768552; AY768553; AY768554;AY768555; AY768556; AY768557; AY768558; AY768559; AY790275; AY790283;AY790297; AY790305; AY790313; AY790314; AY790315; AY790320; AY738451;AY738452; AY738453; AY738454; AY738455; AY738456; AY851460; AY851461;AY862598; AY862599; AY862600; AY862601; AY862602; AY862603; AY862604;AY862605; AY862606; AY937403; AY937404; AY949989; DQ003335; DQ064354;DQ064355; DQ064356; DQ064357; DQ064358; DQ064359; DQ064360; DQ064361;DQ064362; DQ064363; DQ064364; DQ064365; DQ064366; DQ064367; DQ064368;DQ064369; DQ064370; DQ064371; DQ064372; DQ064373; DQ064374; DQ064375;DQ064376; DQ064377; DQ064378; DQ064379; DQ064380; DQ067444; DQ108905;DQ108906; DQ108907; DQ108908; DQ108909; DQ108910; DQ108911; DQ108912;DQ108913; DQ108914; DQ108915; DQ108916; DQ108917; DQ108918; DQ108919;DQ108920; DQ108921; DQ108922; DQ108923; DQ108924; DQ108925; DQ108926;DQ108927; DQ108928; DQ108929; DQ108930; DQ108931; DQ108932; DQ104448;DQ104449; DQ104450; DQ104451; DQ104452; DQ104453; DQ104454; DQ104455;DQ104456; DQ104457; DQ104458; DQ104459; DQ104460; DQ104461; DQ104462;DQ104463; DQ104464; DQ104465; DQ104466; DQ104467; DQ104468; DQ104469;DQ104470; DQ104471; DQ104472; DQ104473; DQ104474; DQ104475; DQ104476;DQ104477; DQ104478; DQ104479; DQ104480; DQ104481; DQ104482; DQ104483;DQ104484; DQ104485; DQ225271; DQ227352; DQ223544; CY004420; CY004642;CY005632; CY005639; CY005746; DQ234277; DQ226106; DQ226107; DQ226108;DQ226109; DQ226110; DQ226111; DQ226112; DQ226113; DQ226114; DQ226115;DQ226116; CY005919; CY005929; CY005934; CY005984; CY005985; CY005986;CY005987; CY005988; CY005989; CY005990; CY005991; CY005992; CY006025;CY006042; CY006018; CY006021; CY006023; DQ299829; DQ299837; DQ299845;DQ299853; DQ299861; DQ390215; DQ464352; DQ473608; DQ473609; DQ473610;DQ473611; DQ473612; DQ473613; DQ473614; DQ465400; DQ485208; DQ485216;DQ485224; DQ681203; DQ681207; DQ681216; DQ681221; DQ885991; DQ787797;DQ787802; CY014613; CY014663; DQ997505; DQ997481; DQ997474; DQ997437;DQ997460; DQ997187; DQ997465; DQ997490; DQ997451; DQ997419; DQ997448;EF070733; EF063510; EF063511; EF063512; EF063513; EF063514; EF063515;EF063516; EF154907; EF154908; EF154909; EF154910; EF154911; EF154912;EF154913; EF154914; EF154915; EF154916; EF154917; EF154918; EF154919;EF154920; EF154921; EF154922; EF154923; EF154924; EF154925; EF154926;EF154927; EF154928; EF154929; EF154930; EF154931; EF154932; EF154933;EF154934; EF154935; EF154936; EF154937; EF154938; EF154939; EF154940;EF154941; EF154942; EF154943; EF154944; EF154945; EF154946; EF154947;EF154948; EF154949; EF154950; EF154951; EF154952; EF154953; EF154954;EF154955; EF154956; EF154957; EF154958; EF154959; EF154960; EF154961;EF154962; EF154963; EF154964; EF154965; EF154966; EF154967; EF154968;EF154969; EF154970; EF154971; EF154972; EF154973; EF154974; EF154975;EF154976; EF154977; EF154978; EF154979; D90305; ABO49159; ABO49160;ABO80224; ABO80225; ABO80226; ABO80227; ABO80228; AB125927; AB125928;AB125929; AB125930; AB125931; AB262463; AB276111; AB256666; AB256674;AB256682; AB256690; AB256698; AB256706; AB256714; AB256722; AB256730;AB256738; AB256746; AB295601; AJ404626; AJ404627; AJ291392; AJ536330;AJ536331; AJ536332; AJ781818; AJ781819; AJ781820; AJ781821; AJ781822;AJ781823; AJ781824; AJ781825; AJ781826; AJ781827; AM087218; AM087219;AM286688 and AM286689.

Polynucleotides encoding influenza virus subtype H10 hemagglutininssuitable for inclusion as a cargo moiety in conjugates herein have thefollowing accession numbers and are available to the public via theNational Center for Biotechnology Information (NCBI) Entrez nucleotidesearch and retrieval system: AB271117; AB274041; CY005996; CY005997;CY005998; CY005999; CY006000; CY006001; CY014619; CY014644; CY014671;CY014739; CY017781; CY020901; CY020909; CY020925; DQ374399; J02110;M21646; M21647; AB289339; AB292412; AB292666; AB292781; AB296078;AF311750; AM087215; AM087216; CY005921; CY005922; CY005930; CY005982;CY005993; CY005994 and CY005995.

Polynucleotides encoding influenza virus subtype H11 hemagglutininssuitable for inclusion as a cargo moiety in conjugates herein have thefollowing accession numbers and are available to the public via theNational Center for Biotechnology Information (NCBI) Entrez nucleotidesearch and retrieval system: AB277756; AB288845; DQ424861; DQ435281;DQ435282; DQ435283; DQ435284; DQ435285; DQ482667; EF200063; J02100;J02106; J02107; J02108; J02161; AB292779; AB292783; AB296076; AB298283;AF310986; AY684895; CY005923; CY005924; CY006002; CY006003; CY006004;CY006005; CY014593; CY014595; CY014679; CY014687; CY014719; CY014806;CY017075; CY017765; CY017845; CY018015; CY020941; CY020949; CY020965;CY021133; CY021141; CY021149; CY021165; CY021173; CY021181; CY021245;CY021253; CY021437; CY021445; CY021469; CY021613; CY021645; CY021653;CY021661; CY021669; CY021685; D90306; DQ080993; DQ327835; DQ424858;DQ424859 and DQ424860.

Polynucleotides encoding influenza virus subtype H12 hemagglutininssuitable for inclusion as a cargo moiety in conjugates herein have thefollowing accession numbers and are available to the public via theNational Center for Biotechnology Information (NCBI) Entrez nucleotidesearch and retrieval system: AB288334; AB288843; AF310990; AF310991;AF310992; AM286685; CY005920; CY005925; CY006006; CY006007; CY006008;CY012840; CY014598; CY014636; CY016419; CY017733; CY017853; CY021293;CY021301; D90307; DQ787811 and J02104.

Polynucleotides encoding influenza virus subtype H13 hemagglutininssuitable for inclusion as a cargo moiety in conjugates herein have thefollowing accession numbers and are available to the public via theNational Center for Biotechnology Information (NCBI) Entrez nucleotidesearch and retrieval system: AB284988; AB285094; AB292664; AM087220;AM087221; AY684886; AY684887; CY005914; CY005931; CY005932; CY005979;CY014603; CY014694; CY014720; D90308; K00383; M26089; M26090 and M26091.

Polynucleotides encoding influenza virus subtype H14 hemagglutininssuitable for inclusion as a cargo moiety in conjugates herein have thefollowing accession numbers and are available to the public via theNational Center for Biotechnology Information (NCBI) Entrez nucleotidesearch and retrieval system: AB289335 and CY014604.

Polynucleotides encoding influenza virus subtype H15 hemagglutininssuitable for inclusion as a cargo moiety in conjugates herein have thefollowing accession numbers and are available to the public via theNational Center for Biotechnology Information (NCBI) Entrez nucleotidesearch and retrieval system: CY006010; CY006033; CY006034; L43917;CY006032; AB295613; CY006009 and L43916.

Polynucleotides encoding influenza virus subtype H16 hemagglutininssuitable for inclusion as a cargo moiety in conjugates herein have thefollowing accession numbers and are available to the public via theNational Center for Biotechnology Information (NCBI) Entrez nucleotidesearch and retrieval system: CY005933; CY014599; CY015160; AY684888;AY684889; AY684890 and AY684891.

Thus, a polynucleotide encoding an influenza virus hemagglutinin may beincluded as a cargo moiety.

In particular embodiments, influenza virus hemagglutinin protein or anantigenic portion thereof is included in a conjugate composition foradministration to a subject to enhance an immune response to influenzavirus.

In other embodiments, the virus conjugated to a sialoadhesin bindingantibody may be used as a gene transfer vector in order to express adesired nucleic acid in a target cell. Such viruses are known in the artand include herpes viruses, adenoviruses and adeno-associated viruses,for example.

In further embodiments, a viral cargo moiety is a virus or portionthereof expressing no non-viral proteins. A cargo moiety virus is aporcine arterivirus in one embodiment.

Conjugation

A cargo moiety is conjugated to a sialoadhesin binding moiety by any ofvarious methods. The conjugation method chosen will depend on thechemical identity of the cargo and the sialoadhesin binding moiety.

A conjugate according to embodiments hereof encompasses a sialoadhesinbinding moiety and a cargo linked together by chemical bonding, covalentor non-covalent, as well as by recombinant techniques includingproduction of a fusion protein, such as a conjugate produced using anucleic acid expression construct encoding a sialoadhesin binding moietyand a cargo.

In particular embodiments, a cargo moiety and a sialoadhesin bindingmoiety are chemically linked via free functional groups on thesemoieties. Such functional groups illustratively include amino, carboxyl,hydroxyl, and sulfhydryl groups.

A linkage between a cargo moiety and a sialoadhesin binding moiety isillustratively an ester, an ether, a carbamate, a carbonate, adisulfide, a peptide, and an amide. The term “linkage” refers to a bondor group formed by chemical reaction between the two moieties such thatthe moieties are covalently coupled, directly or indirectly.

In certain embodiments, a linkage between a sialoadhesin binding moietyand a cargo moiety is labile in an intracellular environment, such thatthe sialoadhesin binding moiety and cargo moiety may be separatedfollowing cell uptake. For instance, a linkage may be susceptible tohydrolysis, enzymatic cleavage, or other form of cleavage, such that thecargo moiety provides a desired effect following such separation fromthe sialoadhesin binding moiety. An ester linkage is one example of alinkage susceptible to hydrolysis in a cell. A disulfide linkage is afurther example of a linkage susceptible to cleavage following celluptake. In other embodiments, a cargo moiety provides a desired effectwhile conjugated to the sialoadhesin binding moiety.

In certain embodiments, more than one cargo moiety may be included in aconjugate composition. Further, more than one sialoadhesin bindingmoiety may be included in a conjugate composition.

Where one or both of the sialoadhesin binding moiety and the cargomoiety include a peptide and/or protein, functional group of a cargomoiety and a sialoadhesin binding moiety used to conjugate thesemoieties can be at N- or C-terminus or at between the termini of one orboth peptides or proteins.

A protective group may be added to a sialoadhesin binding moiety and/orcargo moiety in a process to form a conjugate herein. Such groups, theirgeneration and use are described in Protective Groups in OrganicSynthesis by T. W. Greene and P. G. M. Wuts, John Wiley & Sons, 1999.

Conjugation chemistries used in conjugation of a cargo moiety and asialoadhesin binding moiety illustratively include coupling agents suchas, but not limited to, glutaraldehyde, carbodiimide, succinimideesters, benzidine, periodate, isothionate and combinations of these.

A conjugate herein may be produced using recombinant techniques. Forexample, in particular embodiments, a conjugate is an expression productof a nucleic acid construct including an expression construct encoding afusion protein, the fusion protein including a sialoadhesin bindingmoiety or portion thereof and a cargo moiety linked directly to thesialoadhesin binding moiety or portion thereof or through anintermediate linker.

In particular embodiments, an expression construct encoding a fusionprotein encodes an anti-sialoadhesin antibody or a fragment of ananti-sialoadhesin antibody. Thus, in particular embodiments, anexpression construct encodes a fusion protein including a nucleic acidwhich encodes a cargo moiety and an anti-sialoadhesin antibody orportion thereof. For example, an expression construct encoding a fusionprotein herein encodes a cargo attached to a portion of ananti-sialoadhesin antibody including a variable region of ananti-sialoadhesin antibody such as, but not limited to, a heavy chainvariable region and/or a light chain variable region, a single chainVL-VH region, and/or an H chain C region in particular embodiments.

In particular embodiments, an expression construct encoding a fusionprotein encodes a cargo moiety and mAb 41D3 or a portion of mAb 41D3. Infurther embodiments, an expression construct encoding a fusion proteinencodes an influenza virus hemagglutinin and mAb 41D3 or a portion ofmAb 41D3.

In particular embodiments, an expression construct encoding a fusionprotein encodes a cargo moiety and mAb 7D2 or a portion of mAb 7D2. Infurther embodiments, an expression construct encoding a fusion proteinencodes an influenza virus hemagglutinin and mAb 7D2 or a portion of mAb7D2.

In particular embodiments, an expression construct encoding a fusionprotein encodes a cargo moiety and mAb MCA2316 or a portion of mAbMCA2316. In further embodiments, an expression construct encoding afusion protein encodes an influenza virus hemagglutinin and mAb MCA2316or a portion of mAb MCA2316.

Cloning and expression of nucleic acids encoding antibody regions andfusion proteins including an antibody region are known in the art asexemplified in J. D. Pound (Ed.) Immunochemical Protocols. Methods inMolecular Biology, Humana Press; 2nd ed., 1998, chapter 43; R.Kontermann and S. Dübel (Eds.), Antibody Engineering, Springer LabManuals, Springer, 2001; and B. K. C. Lo (Ed.), Antibody Engineering:Methods and Protocols. Methods in Molecular Biology, Humana Press, 2003.

A cargo moiety and a sialoadhesin binding moiety may be linked directlyto form a conjugate. Alternatively, a linker may be bound to both acargo moiety and to a sialoadhesin binding moiety, such that thesemoieties are indirectly linked through the linker. A linker may be ahomo bifunctional linker or a hetero-bifunctional linker, depending onthe identity of the moieties to be conjugated. Further, a linker may bemultifunctional so as to link more than one cargo moiety and/or morethan one sialoadhesin binding moiety.

In general, a linker has about 1-20 backbone carbon atoms. However, alinker may be larger or smaller.

Optionally, a linker is encoded by a nucleic acid in an expressionconstruct.

A linker may be a natural or synthetic polymer in some embodiments. Forexample, suitable polymers include agarose, carboxymethylcellulose,cellulose, dextran, and polyaminopolystyrene. A preferred polymer ispolyacrylamide, PEO (polyethylene) or PEG (polyethylene glycol) spacer.

In certain embodiments, a sialoadhesin binding moiety including a sialicacid and/or sialylated structure may be conjugated to a cargo moietydirectly or indirectly. For example, a sialic acid residue may beconjugated to a lipid-containing cargo moiety to form a glycolipidconjugate composition and/or to a protein or peptide cargo moiety byN-linkage or O-linkage to form a glycopeptide or glycoprotein conjugateherein. A sialic acid residue may also be conjugated to a linker.

Pharmaceutical Compositions and Administration

A conjugate can be administered to a subject alone or as part of apharmaceutical composition. Conjugate compositions are suitable foradministration to patients by a variety of routes illustrativelyincluding, but not limited to, intravenous, oral, parenteral,intramuscular, subcutaneous and mucosal.

The pharmaceutical compositions hereof include a conjugate and apharmaceutically acceptable carrier. The term “pharmaceuticallyacceptable” refers to a material which can be administered to a subjectalong with a conjugate composition without causing significantundesirable biological effects and without interacting in a deleteriousmanner with any other component of the pharmaceutical composition.Pharmaceutical compositions suitable for administration illustrativelyinclude physiologically acceptable sterile aqueous or nonaqueoussolutions, dispersions, suspensions or emulsions, and sterile powdersfor reconstitution into sterile injectable solutions or dispersions.Examples of suitable aqueous and nonaqueous carriers; diluents;solvents; or vehicles include water, ethanol, polyols such as, but notlimited to, propylene glycol, polyethylene glycol, glycerol, and thelike, suitable mixtures thereof; vegetable oils such as, but not limitedto, olive oil; and injectable organic esters such as, but not limitedto, ethyloleate. Proper fluidity can be maintained, e.g., by the use ofa coating such as, but not limited to, lecithin, by the maintenance ofthe required particle size in the case of dispersions, and by the use ofsurfactants.

Compositions suitable for injection optionally include physiologicallyacceptable sterile aqueous or nonaqueous solutions, dispersions,suspensions or emulsions, and sterile powders for reconstitution intosterile injectable solutions or dispersions. Examples of suitableaqueous and nonaqueous carriers, diluents, solvents or vehicles includewater, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol,and the like), suitable mixtures thereof, vegetable oils (such as oliveoil) and injectable organic esters such as, but not limited to, ethyloleate. Proper fluidity can be maintained, e.g., by the use of a coatingsuch as, but not limited to, lecithin, by the maintenance of therequired particle size in the case of dispersions and by the use ofsurfactants.

Pharmaceutical compositions herein may also contain adjuvants such as,but not limited to, preserving, wetting, emulsifying, and dispensingagents. Prevention of the action of microorganisms can be ensured byvarious antibacterial and antifungal agents, e.g., parabens,chlorobutanol, phenol, sorbic acid, and the like. It may also bedesirable to include isotonic agents, e.g., sugars, sodium chloride, andthe like. Prolonged absorption of an injectable pharmaceutical form canbe brought about by the use of agents delaying absorption, e.g.,aluminum monostearate and gelatin.

Further exemplary adjuvants include immunostimulating adjuvants such as,but not limited to, Freund's complete adjuvant; Freund's incompleteadjuvant; aluminum hydroxide such as commercially available asAlhydrogel, Accurate Chemical & Scientific Co, Westbury, N.Y.; and Gerbuadjuvant, available from C—C Biotech, Poway, Calif.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, a conjugate isadmixed with at least one inert customary excipient (or carrier) suchas, but not limited to, sodium citrate or dicalcium phosphate or (a)fillers or extenders, as for example, starches, lactose, sucrose,glucose, mannitol, and silicic acid, (b) binders, as for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone,sucrose, and acacia, (c) humectants, as for example, glycerol, (d)disintegrating agents, as for example, agar-agar, calcium carbonate,potato or tapioca starch, alginic acid, certain complex silicates, andsodium carbonate, (e) solution retarders, as for example, paraffin, (f)absorption accelerators, as for example, quaternary ammonium compounds,(g) wetting agents, as for example, cetyl alcohol, and glycerolmonostearate, (h) adsorbents, as for example, kaolin and bentonite, and(i) lubricants, as for example, talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, or mixturesthereof. In the case of capsules, tablets, and pills, the dosage formsmay also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethyleneglycols, andthe like.

Solid dosage forms such as, but not limited to, tablets, dragees,capsules, pills, and granules can be prepared with coatings and shells,such as, but not limited to, enteric coatings and others well known inthe art. They may contain opacifying agents, and can also be of suchcomposition that they release the active compound or compounds in acertain part of the intestinal tract in a delayed manner.Microencapsulated formulations of a conjugate are also contemplated.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirs. Inaddition to a conjugate herein, the liquid dosage forms may containinert diluents commonly used in the art, such as, but not limited to,water or other solvents, solubilizing agents and emulsifiers, as forexample, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl alcohol, benzyl benzoate,propyleneglycol, 1,3-butyleneglycol, dimethylformamide, oils, inparticular, cottonseed oil, groundnut oil, corn germ oil, olive oil,castor oil and sesame oil, glycerol, tetrahydrofurfuryl alcohol,polyethyleneglycols and fatty acid esters of sorbitan or mixtures ofthese substances, and the like.

Besides such inert diluents, a pharmaceutical composition herein canalso include adjuvants, such as, but not limited to, wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Suspensions, in addition to a conjugate, may contain suspending agents,e.g., ethoxylated isostearyl alcohols, polyoxyethylene sorbitol andsorbitan esters, microcrystalline cellulose, aluminum metahydroxide,bentonite, agar-agar and tragacanth, or mixtures of these substances,and the like.

Further specific details of pharmaceutical formulation can be found inPharmaceutical Dosage Forms Tablets, eds. H. A. Lieberman et al., NewYork: Marcel Dekker, Inc., 1989; L. V. Allen, Jr. et al., Ansel'sPharmaceutical Dosage Forms and Drug Delivery Systems, 8th Ed.,Philadelphia, Pa., Lippincott, Williams & Wilkins, 2004; and Remington,The Science and Practice of Pharmacy, 21^(st) ed., Lippincott, Williams& Wilkins, Philadelphia, Pa., 2006.

A conjugate may be delivered in conjunction with a non-conjugatedtherapeutic and/or diagnostic agent in one embodiment. A therapeuticand/or diagnostic agent suitable in this regard illustratively includesan analgesic, an antibiotic, an antibody, an antigen, ananti-inflammatory, an anti-tumoral agent, an antiviral, a gamma or betaradiation emitting species, an enzyme, and a hormone. In addition, twoor more conjugate compositions may be administered to a subject.

The dosage of an inventive pharmaceutical composition will vary based onfactors such as, but not limited to, the route of administration; theage, health, and weight of the subject to whom the composition is to beadministered; the nature and extent of the subject's symptoms, if any,and the effect desired. Usually a daily dosage of a conjugate is in therange of about 0.001 to 100 milligrams per kilogram of a subject's bodyweight. A daily dose may be administered as two or more divided doses toobtain the desired effect. An inventive pharmaceutical composition mayalso be formulated for sustained release to obtain desired results.

For example, a parenteral composition suitable for administration byinjection includes 1% by weight of a conjugate in buffered saline.

Methods

A method of delivering a cargo moiety to a cell is provided whichincludes contacting a cell expressing sialoadhesin with a conjugateherein. The sialoadhesin binding moiety present in the conjugate bindsto the sialoadhesin expressed by the cell and the conjugate isinternalized in the cell. The cell may be in vivo, ex vivo or in vitro.

Sialoadhesin is expressed primarily by macrophages. Thus, in oneembodiment of an inventive method, a drug delivery system targetingmacrophages is provided. Thus, in such an embodiment, a cell contactedwith a conjugated sialoadhesin binding moiety and cargo moiety is amacrophage.

A cell contacted with a conjugate composition in a method hereinexpresses sialoadhesin naturally or may be induced to do so. In such amethod, cells other than macrophages may be targeted.

For example, a cell may be transfected with an expression constructencoding sialoadhesin such that sialoadhesin is expressed in the cell.An expression construct includes a nucleic acid encoding full-lengthsialoadhesin, or a portion thereof, operably linked to a regulatoryelement. Full-length nucleic acids encoding sialoadhesin have beenisolated from various species and exemplary polynucleotides and encodedsialoadhesin proteins are described herein. A regulatory elementoperably linked to the nucleic acid encoding sialoadhesin illustrativelyincludes a promoter, an enhancer, an origin of replication, apolyadenylation signal, and a transcription termination sequence.Expression constructs and methods for their generation are known in theart, as described, e.g., in Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, 2001; and F.Ausubel et al. (Eds.), Short Protocols in Molecular Biology, Wiley,2002.

In particular embodiments, an expression construct encoding asialoadhesin protein encodes the sialoadhesin protein of SEQ ID NO:5,SEQ ID NO:7, SEQ ID NO:9, or a biologically active homologue thereof. Inparticular embodiments, an expression construct encoding a sialoadhesinprotein includes a nucleotide sequence of SEQ ID NO:6, SEQ ID NO:8, orSEQ ID NO:10.

Biological activity of a putative sialoadhesin homologue may bedetermined by one of skill in the art, e.g., by using a functionalassays described herein or other functional assays known in the art.

An expression construct encoding sialoadhesin is generated according tomethods known in the art. For example, a pcDNA3.1/Sn plasmid containingthe porcine sialoadhesin cDNA cloned into the pcDNA3.1 vector(Invitrogen) described in N. Vanderheijden et al., 2003, J. Virol.77:8207-15 is a sialoadhesin expression construct.

A cell transfected with an expression construct to induce or enhancesialoadhesin expression in the cell may be transiently transfected inparticular embodiments. Alternatively, a stable cell line expressingsialoadhesin is produced.

Any of various cells may be used to produce a cell line stablyexpressing sialoadhesin, e.g., including, but not limited to THP-1cells, PK-15 cells, 3D4/31 cells, and HEK293T cells.

Methods of producing a stable cell line expressing a desired protein areknown in the art, e.g., in standard molecular biology references such asS. Ozturk and W.-S. Hu (Eds.), Cell Culture Technology forPharmaceutical and Cell-Based Therapies. Biotechnology and BioprocessingSeries, CRC Press, 2005.

Briefly described, cells are transfected with an expression constructencoding sialoadhesin. For example, cells are transfected with anexpression construct including SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10or another sequence encoding SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9 ora homologue thereof. A transfected expression construct further encodesresistance to a selection agent, including, but not limited to,resistance to neomycin (G418). Expression constructs conferringresistance to a selection agent are known in the art and arecommercially available or may be constructed using standard molecularbiology techniques.

Cells are transfected according to standard transfection methodsillustratively including, but not limited to, calcium phosphatetechniques and lipofectin techniques such as described in Sambrook etal., Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press, 2001; and F. Ausubel et al. (Eds.), Short Protocols inMolecular Biology, Wiley, 2002.

Following transfection, cells are incubated, on cell culture plates orin cell culture wells for instance, in medium containing a selectionagent, such as 0.5 g/L neomycin. Cells not transfected or not expressingthe resistance marker die following incubation with the selection agent,generally after several days. Dead cells are removed from the vicinityof living transfected cells in order to select for particular clones.Transfected cells are typically disposed individually, in wells or incloning cylinders for example, in order to select one or more stablytransfected cell lines. Once individual colonies have grown, they can beassayed for sialoadhesin expression, such as by ELISA. Stablytransfected cells are further assayed for binding of a sialoadhesinbinding moiety and/or conjugate and uptake of the binding moiety and/orconjugate into the cell.

Stably transfected cells may be used in methods hereof. For example, astable cell line expressing sialoadhesin is used in a method hereof fortransfection of a cell by delivery of a conjugate including asialoadhesin binding moiety and a nucleic acid.

In further embodiments of methods herein, a cell is treated with anagent effective to induce or enhance expression of sialoadhesin in thecell. In particular embodiments, a cell is treated with a cytokineeffective to induce or enhance expression of sialoadhesin in the cell.For example, a cell treated with a cytokine effective to induce orenhance expression of sialoadhesin is a monocyte, a monocyte cell line,a macrophage and a macrophage cell line.

In particular embodiments, a human cell and/or a human-derived cell lineis treated with a cytokine effective to induce or enhance expression ofsialoadhesin. An example of a human-derived cell line is human monocytecell line THP-1. A suitable cytokine effective to induce or enhanceexpression of sialoadhesin is interferon-alpha (INF-alpha).

Human monocytes are treated with INF-alpha to induce or enhanceexpression of sialoadhesin in a particular embodiment. The monocytes maybe isolated, for instance from blood, and treated in vitro withINF-alpha. Sialoadhesin expression may be assessed by assaysillustratively including, but not limited to, immunoassay.

In further embodiments, an effective amount of INF-alpha is administeredto a subject such that sialoadhesin expression is induced or enhanced incells in vivo. An effective amount is illustratively between 10 to 500units IFN-alpha per ml of blood of the subject.

Methods are provided for transfection of a cell using a conjugate hereinincluding a cargo nucleic acid, particularly a cargo expressionconstruct. Cells expressing sialoadhesin are contacted with a conjugateincluding a sialoadhesin binding moiety and a cargo expression constructin a particular embodiment in order to express an encoded protein orpeptide in the cells. Transfection using a sialoadhesin binding moietyand a cargo expression construct is used in sialoadhesin expressingcells in vitro or in vivo. Transfection using a conjugate providedherein is useful to increase the level of a desired protein or peptidein a cell, for instance, to produce recombinantly expressed protein, forinstance, to study function of the protein.

A method includes contacting a cell expressing sialoadhesin with aconjugate composition may be used to stimulate an immune response in asubject, for instance, to vaccinate the subject.

Vaccination is one of the earliest used and most powerful tools forstimulating an organism to defend against infection. Broadly described,vaccination is a method of administering an antigen to an organism inorder to stimulate the organism's immune system to provide a cellularand/or molecular defensive response.

While vaccination by non-cell targeted administration of an antigen toan organism can be effective, in some cases large amounts of antigenmust be administered in order to achieve a desired response. Further, anon-cell targeted administration may require a longer time and/or morebooster administrations of the antigen to achieve an effective immuneresponse. Thus, compositions and methods for stimulating an immuneresponse in a subject are needed. Such a method is provided herein andincludes administering to a subject an effective amount of a conjugatecomposition herein which includes a sialoadhesin binding moietyconjugated to an antigen. An immune response may be stimulated in orderto inhibit infection by a pathogen, or to stimulate an antitumoralresponse for instance.

An immune response may be measured, e.g., by assay of the subject'sserum for antibodies to an antigen administered as part of a conjugate.Applicable immunoassays include, e.g., ELISA performed on a samplebefore and at one or more times following administration of theconjugate.

In certain embodiments, administration of a composition effective totarget an antigen to an antigen presenting cell, particularly amacrophage, is included in a method provided herein.

In a specific example, vaccination of swine against PRRSV is anembodiment hereof. PRRSV is an infectious disease of swine which cancause severe respiratory disorders, as well as abortion. The viral agenthas been identified, as described in G. Weensvoort et al., 1991,Veterinary Review 13:121-130. However, there is currently no effectivetreatment for this disease which can frequently only be controlled bydestruction of the herd, resulting in considerable cost to swineproducers.

A vaccine and method for vaccination of a pig against PRRSV is provided.A conjugate composition including a sialoadhesin binding moiety whichbinds to porcine sialoadhesin is conjugated to a PRRSV, a PRRS protein,or an antigenic portion of a PRRSV or protein. The conjugate compositionis administered to a pig in an amount effective to stimulate an immuneresponse. The route of administration may be any convenient route,illustratively including, but not limited to, intravenous,intramuscular, intraperitoneal, subcutaneous, oral, mucosal, and anycombination thereof.

In a further example, vaccination of a subject against an influenzavirus is an embodiment hereof. Influenza virus is an infectious diseaseof numerous species which can cause severe respiratory symptoms anddeath.

A vaccine and method for vaccination of a subject against influenzavirus is provided. A conjugate composition including a sialoadhesinbinding moiety which binds to sialoadhesin is conjugated to an influenzavirus, an influenza virus protein or an antigenic portion of aninfluenza virus or protein. In particular embodiments, compositions andmethods for vaccination of a subject against a type A influenza virusare provided. The conjugate composition is administered to a subject inan amount effective to stimulate an immune response against influenzavirus. The route of administration may be any convenient route,illustratively including, but not limited to, intravenous,intramuscular, intraperitoneal, subcutaneous, oral, mucosal, and anycombination thereof.

In specific embodiments, compositions and methods for vaccination of aporcine subject against an influenza virus are provided. Inventivemethods and compositions for vaccination against influenza virus are notlimited to porcine subjects and may be used in other subjectssusceptible to influenza virus infection, illustratively including, butnot limited to, humans and birds.

A conjugate composition for vaccination of a subject against aninfluenza virus includes a sialoadhesin binding moiety and an influenzavirus hemagglutinin protein or antigenic portion thereof in particularembodiments. In a specific example, a conjugate composition forvaccination of a subject against an influenza virus includes the proteinencoded by SEQ ID NO:3 or a homologue thereof. In a further specificexample, a conjugate composition for vaccination of a subject against aninfluenza virus includes the protein identified as SEQ ID NO:4 or ahomologue thereof.

Traditionally, achieving desired antibody titers can be difficult withsome antigens, such as inactivated or subunit vaccines, requiringmultiple administrations of the antigen. Targeted delivery of an antigento sialoadhesin expressing macrophages using an inventive compositionincluding antigen coupled to a sialoadhesin-specific mAb allowsincreased titers of antigen-specific antibodies. Targeted deliveryelicits an immune response which is more efficient in comparison toadministration of an unconjugated antigen, since antibodies appearearlier after administration and higher titers are reached.

Thus, in certain embodiments, a method for stimulating the immune systemof a subject includes a single administration of a conjugate compositionhaving an antigen cargo moiety hereof. Additional administrations ofsuch a conjugate may be performed in alternative embodiment hereof.

The term “subject” refers to a vertebrate to which a conjugate is to beadministered. A subject is preferably a mammal, and more preferably ahuman in particular embodiments. In further embodiments, a preferredsubject is porcine. However, the term subject is not limited to eitherhuman or porcine subjects and methods and compositions hereof may beused in conjunction with any of various animals illustratively includingcows, horses, chickens and other poultry, goats, rodents, cats, dogs andbirds.

An effective amount is an amount sufficient to achieve an intendedbeneficial or desired result. In general, an effective amount is in therange of about 0.001 to 100 milligrams per kilogram of a subject's bodyweight.

In a further embodiment hereof, a cell expressing sialoadhesin istargeted in order to eliminate or inhibit the cell. For example,elimination or inhibition of sialoadhesin expressing macrophages isdesirable in certain disease states, such as, but not limited to,rheumatoid arthritis.

Another embodiment hereof relates to delivery of a therapeutic agent toinhibit pathogenic infection. Thus, one embodiment of an inventivemethod includes targeted delivery to macrophages of a conjugatecomposition herein including an antimicrobial drug cargo moiety. Suchtargeted delivery allows the use of antimicrobial drugs that haveundesirable side effects when a non-targeted delivery system is used,such as systemic administration of free antimicrobial drug.

The following illustrative examples further describe the invention.

EXAMPLES Example 1

An assay for assessment of binding of a sialoadhesin binding moiety tosialoadhesin is described in this example along with an assay forassessing uptake of the bound sialoadhesin binding moiety into a cell.

In this example, primary porcine alveolar macrophages, cells whichexpress sialoadhesin, are used to assess binding and/or uptake of asialoadhesin binding moiety.

Porcine alveolar macrophages are isolated from 4- to 6-week oldconventional Belgian Landrace pigs from a PRRSV negative herd asdescribed in G. C. Wensvoort et al., 1991, Vet Q 13:121-30. Briefly, themain bronchus of each lung half was clamped and a needle was inserteddistally. Cold PBS (3×20 ml) was injected, followed by massage of thelung tissue and aspiration. About 75% of the BAL fluid could beaspirated and was kept on ice. BAL cells were separated from fluids bycentrifugation and cells were used in the experiments. Staining with mAb41D3 showed that this procedure routinely resulted in a purity of morethan 95% of sialoadhesin expressing macrophages.

The cells are cultivated in Earle's MEM, supplemented with 10% fetalbovine serum (FBS), 2 mM L-glutamine (BDH Chemicals Ltd.), 1%non-essential amino acids (Gibco BRL), 1 mM sodium pyruvate andantibiotics in a humidified 5% CO2 atmosphere at 37° C. Macrophages arepreferably cultivated for 24 hours before use.

Control cells, such as non-sialoadhesin-expressing cells, may be used toassess specificity of binding and uptake. Such cells include, e.g.,HEK293T cells, a human embryonic kidney cell line transfected with SV40large T-Ag (SV40TtsA1609) described in RB. DuBridge et al., 1987, Mol.Cell. Biol. 7:379-8. HEK293T cells are maintained in DMEM supplementedwith 10% FBS, 2 mM L-glutamine and a mixture of antibiotics.

Antibodies used in this example include mAb 41D3 directed againstsialoadhesin. Control antibodies include isotype matched (IgG1) mAb13D12, directed against PRV glycoprotein gD described further in H. J.Nauwynck and M. B. Pensaert, 1995, Arch. Virol. 140:1137-46; and mAb74-22-15, reactive with SWC3, a membrane/surface protein used as amarker of porcine monocytes, macrophages and neutrophils described in M.D. Pescovitz et al., 1984, J. Immunol. 133:368-75.

Antibodies are purified using protein G sepharose column chromatography(Amersham Biosciences), dialyzed to PBS and stored at 4° C. or −70° C.prior to use.

In an assay to assess characteristics of a sialoadhesin binding moiety,cells are incubated with a sialoadhesin binding moiety under variousconditions and at various concentrations. In this example, primarymacrophages are incubated with purified antibodies at a concentration of25 μg/ml for 1 hour at 4° C. to allow only attachment, but nointernalization. Cells are then washed to remove unbound antibody andshifted to 37° C. to start endocytosis. After different times, cells arefixed with 3% paraformaldehyde (PF), permeabilized with 0.1% TRITON®X-100, and stained with FITC-labeled goat-anti-mouse IgG to visualizeantibodies bound to and internalized in the cells. As a control, cellsare fixed after the 4° C. incubation (time 0). The number of vesiclesinternalized in the macrophages and control cells incubated undervarious conditions may be counted using an appropriate technique, e.g.,confocal microscopy.

Confocal analysis is performed using a scanning spectral confocalsystem, such as a Leica TCS SP2 laser linked to a Leica DM IRBE invertedmicroscope, from Leica Microsystems GmbH. Image acquisition is performedusing a Leica TCS SP2 confocal software package and overlay images areproduced with Adobe Photoshop CS.

In a particular example, macrophages are incubated for 60 minutes at 4°C. with the sialoadhesin-specific mAb 41D3 to allow antibody binding,but no internalization. Cells are then washed to remove unboundantibody, and shifted to 37° C. to allow internalization. Cells arefixed and stained at different times for analysis of binding and uptakeinto cells.

FIG. 1 shows that incubation of primary porcine macrophages with mAb41D3 induces sialoadhesin and antibody internalization. FIG. 1 is agraph illustrating specific binding and internalization of asialoadhesin binding moiety at different times after incubation ofmacrophages at 37° C. with mAb 41D3. Kinetics of uptake are demonstratedby the percentage of cells with internalized sialoadhesin at differenttimes after incubation of macrophages at 37° C. with mAb 41D3. Data inFIG. 1 represent the means±standard deviations of three independentexperiments. At time 0, a clear membrane staining is observed, and noneof the macrophages contain sialoadhesin positive vesicles in thecytoplasm, as indicated by the point at the origin of the graph.

With increasing time at 37° C., the number of cells which internalizedsialoadhesin and antibody increases to a maximum of 90% at 90 minutesafter the 37° C. shift (FIG. 1), and then declines to 61% at 120 minutesand 50% at 180 minutes. At early time points, endocytic vesicles aremainly present in the vicinity of the plasma membrane, while withincreasing time, endocytosed sialoadhesin is mainly localized to theperinuclear region. As a control, primary porcine macrophages areincubated with a non-sialoadhesin binding antibody, isotype matched mAb13D12, or mAb 74-22-15. Cells incubated with mAb 13D12 show no staining(data not shown), while mAb 74-22-15 incubated cells show exclusiveplasma membrane staining at all time points examined. Further, when mAb41D3 is added to macrophages directly at 37° C., this results in similarinternalization kinetics.

Example 2

Various cells may be used in an assay to assess uptake and/orinternalization of a sialoadhesin binding moiety. A cell line expressingsialoadhesin may be used for assay of binding and/or uptake of asialoadhesin binding moiety.

A porcine cell line, PK-15, may be used in an assay to assess uptakeand/or internalization of a sialoadhesin binding moiety. PK-15 cells aremaintained as described by N. Vanderheijden et al., 2003, J. Virol.77:8207-15. About 25% of PK-15 cells usually express sialoadhesin. PK-15cells are optionally transfected with a sialoadhesin expressionconstruct to enhance expression of sialoadhesin in the cells for use inan assay to assess uptake and/or internalization of a sialoadhesinbinding moiety.

The porcine alveolar macrophage cell line 3D4/31 (37) is maintained inRPMI/MEM (50/50) supplemented with 10% FBS, 2 mM L-glutamine, 1%non-essential amino acids (Gibco) and a mixture of antibiotics. About 5%of 3D4/31 cells usually express sialoadhesin. 3D4/31 are optionallytransfected with a sialoadhesin expression construct to enhanceexpression of sialoadhesin in the cells for use in an assay to assessuptake and/or internalization of a sialoadhesin binding moiety.

HEK293T cells are transfected using calcium phosphate (Cellphecttransfection kit, Amersham Biosciences), and PK-15 and 3D4 cells aretransfected using Lipofectamine Plus (Invitrogen), following themanufacturers' instructions. Cells are used for experiments 24 hoursafter transfection.

Example 3

A primary cell or cell line characterized by little or no expression ofsialoadhesin may be treated to express sialoadhesin and/or to enhancesialoadhesin expression. In particular embodiments, a cell istransfected with a sialoadhesin expression construct in order to providea cell used in an assay for assessment of binding and/or uptake of asialoadhesin binding moiety. An expression construct including anucleotide sequence encoding pig, mouse or human sialoadhesin detailedherein may be used. A pcDNA3.1/Sn plasmid containing the porcinesialoadhesin cDNA cloned into the pcDNA3.1 vector (Invitrogen) isdescribed in N. Vanderheijden et al., 2003, J. Virol. 77:8207-15.

Example 4

In further embodiments, a cell is treated with a stimulator ofsialoadhesin expression in order to provide a cell used in an assay forassessment of binding and/or uptake of a sialoadhesin binding moiety.Stimulators of sialoadhesin expression include interferon-alpha. In thisexample, human peripheral blood mononuclear cells (PBMC) are isolatedfrom heparinized blood from a healthy donor via centrifugation onFicoll-paque according to the manufacturer's instructions (AmershamBiosciences). Monocytes are semi-purified by plastic adhesion andseveral washing steps to remove non-adherent lymphocytes. Flowcytometric analysis with a mouse-anti-human CD14 antibody shows thatthis procedure routinely results in a purity of the monocytes of >90%.Cells are cultivated for 3 days in RPMI medium with 10% FBS (RPMI-FBS)or RPMI-FBS with interferon-gamma, 500 U/ml and Tumor NecrosisFactor-alpha (TNF-alpha), 10 ng/ml, as described in A. Hartnell et al.,Blood, 2001. 97(1): p. 288-96, or in RPMI-FBS supplemented withinterferon-alpha (100 U/ml).

Cells are lifted from a plastic substrate to which they have adhered byincubation with ice-cold PBS for 30 minutes at 4° C. Cells are firstincubated at 4° C. with a mouse anti-human-sialoadhesin specificantibody, 7D2, or a isotype-matched irrelevant control antibody, 13D12.Next, cells are fixed with paraformaldehyde (3% in PBS) or incubated at37° C. for 1 hour for the internalization of the bound antibodiesfollowed by paraformaldehyde fixation. Cells are washed three times andsubsequently incubated with FITC-labeled goat-anti-mouse Ab (MolecularProbes). Some of the cells are double stained with APC-labeledmouse-anti-human CD14 (BD Pharmingen). Finally, the cells are washed twotimes, resuspended in PBS and analyzed with a Becton-Dickinson (SanJose, Calif.) FACScalibur. Ten thousand cells are analyzed for eachsample, and four parameters are stored for further analysis: forwardlight scatter, sideward light scatter, green and red fluorescence, andresults of this analysis are shown in FIG. 5.

Flow cytometric analysis of sialoadhesin expression yields datarepresentative of three experiments. The control sample (shown at I inFIG. 5) is treated as the others during staining but without antibodies.Untreated and cytokine treated cells are stained with a control antibody13D12 and with a human sialoadhesin-specific antibody 7D2, shown at IIin FIG. 5. After the binding of 7D2, one sample was incubated for 1 hourat 37° C. to enable the receptor, sialoadhesin, to internalize theantibodies, shown at III in FIG. 5. IFN-alpha treatment clearly inducesSn expression and the induced Sn is able to internalize monoclonalantibody 7D2. Internalization of monoclonal antibody 7D2 is demonstratedby the reduction in the median fluorescence intensity upon surfacestaining of interferon-alpha treated cells incubated at 37° C. with FITClabeled goat-anti-mouse IgG (Molecular Probes, Invitrogen), as shown inFIG. 5.

In two of the three experiments using human monocytes, low levels ofsialoadhesin is present on the untreated cells, in the third experimentit is absent. Treatment of the cells with TNF-alpha and IFN-gammainduces Sn expression; however, treatment with IFN-alpha leads to asignificantly higher expression of sialoadhesin. Similar results areobtained using monocytes isolated from peripheral blood from pigs andtreated with IFN-alpha to induce Sn expression.

Sialoadhesin induced by IFN-alpha treatment is biologically active asshown by sialic acid binding capacity of IFN-alpha treated monocytes.Red blood cells contain sialic acids on their surface which allows themto bind to monocytes if these have functional expression ofsialoadhesin. Monocytes are grown in 96-well plates for three days asdescribed above. Next, they are incubated for 30 minutes at 37° C. withnormal medium or medium supplemented with neuraminidase (Roche) 30 U/mlto remove sialic acids present on the surface. After removal of theneuraminidase, monocytes are incubated for 1 hour at room temperaturewith a 0.1% solution of human erythrocytes. Excess erythrocytes arewashed away and binding of red blood cells to sialoadhesin is visualizedvia light microscopy. When sialic acids present on the surface of themonocytes are not removed, red blood cells are unable to bind to themonocytes under any conditions tested. However when sialic acids areremoved from monocytes, red blood cells are able to bind in someconditions. Little binding is observed in cells grown in normal medium.Cells treated with TNF-alpha and IFN-gamma do not bind RBC. However, inthe IFN-alpha treated cells clear formation of rosettes, that is, redblood cells bound to monocytes are observed. These data confirm theresults obtained in the flow-cytometric analysis showing thatbiologically active sialoadhesin is induced in monocytes by IFN-alphatreatment.

The ability of cytokine-induced sialoadhesin expression on humanmonocytes to internalize a sialoadhesin binding moiety is also shown inthis example. Human monocytes are isolated as described above andtreated with IFN-alpha for three days to induce human sialoadhesin.Cells are then incubated with human sialoadhesin-specific mAb 7D2 for 60minutes at 37° C. to allow binding and internalization. As a control,the cells are incubated with mAb 7D2 at 4° C. At 4° C., cells are nolonger capable of mediating internalization, thus this control shouldonly binding of the sialoadhesin binding moiety mAb 7D2. After 60minutes, the cells are fixed with 3% paraformaldehyde in PBS andpermeabilized by incubation with 0.1% Triton X-100 in PBS for 2 minutes.MAb 7D2 is visualized by incubation with FITC-labeled goat-anti-mouse(Invitrogen). Cortical actin is also visualized, using TexasRed labeledPhalloidin, to allow discrimination of surface bound and internalizedsialoadhesin. Surface expression of sialoadhesin and binding ofsialoadhesin binding moiety mAb 7D2 is observed at time 0. Followingincubation for 60 minutes at 37° C., internalized sialoadhesin andsialoadhesin binding moiety mAb 7D2 is observed in the IFN-alpha treatedhuman monocytes.

Thus, an in vitro system for evaluation of human sialoadhesin bindingmoieties and conjugates of human sialoadhesin binding moieties isprovided which is analogous to the in vitro and in vivo pig system forevaluation of sialoadhesin binding moieties and conjugates ofsialoadhesin binding moieties.

Example 5

The effect of interferon-alpha on sialoadhesin expression in human THP-1cells, a monocytic continuous cell line, is tested to determine ifsialoadhesin is internalized in these cells upon stimulation with anantibody as a sialoadhesin binding moiety. THP-1 cells are depositedwith the American Type Culture Collection (ATCC) and are identified byATCC Number TIB-202. THP-1 cells are cultivated for 3 days in RPMImedium with 10% FBS (RPMI-FBS) or RPMI-FBS with interferon-gamma (500U/ml) and TNF-alpha (10 ng/ml) or in RPMI-FBS supplemented withinterferon-alpha (100 U/ml).

THP-1 cells are incubated at 4° C. with a human-sialoadhesin specificantibody, 7D2, or an isotype-matched irrelevant control antibody, 13D12.Next, cells are fixed with paraformaldehyde (3% in PBS) or incubated at37° C. for 1 hour to allow antibody induced internalization ofsialoadhesin and the bound antibody followed by paraformaldehydefixation and permeabilization of the cells with 0.1% Triton X-100. Cellsare washed 3 times and subsequently incubated with FITC-labeledgoat-anti-mouse Ab (Molecular Probes). Some of the cells are doublestained with APC-labeled mouse-anti-human CD14 (BD Pharmingen). Thecells are washed two times, resuspended in PBS and analyzed with aBecton-Dickinson (San Jose, Calif.) FACScalibur. Ten thousand cells areanalyzed for each sample, and four parameters were stored for furtheranalysis: forward light scatter, sideward light scatter, green and redfluorescence (FIG. 10). These data show that IFN-alpha treatment induceshuman sialoadhesin on THP-1 cells, and that upon stimulation with mAb7D2 at 37° C., a decrease in cell surface sialoadhesin fluorescence isobserved, indicative of internalization of the antibody bound tosialoadhesin.

FIG. 10 shows flow cytometric analysis of sialoadhesin expression andantibody induced sialoadhesin internalization. Histograms arerepresentative for three experiments. The control sample (I) is treatedas the others during staining but without antibodies. Untreated andcytokine treated cells are stained with a control antibody 13D12 andwith a human sialoadhesin-specific antibody 7D2 (II). After the bindingof 7D2, one sample was incubated for 1 hour at 37° C. to enable thereceptor to internalize the antibodies (III). IFN-alpha treatmentclearly induces sialoadhesin expression and the induced sialoadhesin isable to internalize monoclonal antibody 7D2 as shown by the decreasedmedian which lowers from 364 to 258 upon incubation at 37° C.

Confocal microscopy is used in this example to visualize internalizationof sialoadhesin and bound sialoadhesin binding moiety mAb 7D2. THP-1cells are incubated with human sialoadhesin-specific mAb 7D2 for 60minutes at 37° C. to allow internalization. As a control, a time 0 wasanalyzed by incubating the cells with mAb 7D2 at 4° C. At 4° C., cellsare no longer capable of mediating internalization, thus this controlshould only show binding to sialoadhesin at the cell surface withoutinternalization. After 60 minutes, the cells are fixed with 3%paraformaldehyde in PBS and permeabilized by incubation with 0.1% TritonX-100 in PBS for 2 minutes. Internalized antibodies are visualized byincubation and staining with FITC-labeled goat-anti-mouse (Invitrogen).Surface labeling of these cells is observed at time 0, while at time 60,sialoadhesin and bound antibody is observed internalized in the THP-1cells.

Thus, an in vitro system is provided including the human monocytic THP-1cell line, allowing further analysis of antibody-induced humansialoadhesin internalization without the need of isolating primary bloodmonocytes or macrophages.

Example 6

Chemical cross-linking of a sialoadhesin binding moiety and a cargomoiety is described. In this example, human serum albumin (HSA) is acargo moiety which is an antigen to be conjugated to mAb 41D3, asialoadhesin binding moiety, to form a conjugate composition. Inaddition, as a control, human serum albumin (HSA) is conjugated to anon-sialoadhesin binding antibody, mAb 13D12.

For chemical cross-linking of HSA and the mAb in this example, a twostep cross-linking protocol is used. The amine reactive cross-linkerLC-SMCC (Pierce) is coupled to the purified mAb 41D3 by incubating 600micrograms of LC-SMCC with 20 milligrams of mAb in 8 millilitersphosphate buffered saline (PBS) for 30 minutes at room temperature. Theamine-reactive cross-linker SPDP (Pierce) is coupled to the purified HSAby incubating 2 milligrams SPDP with 40 milligrams HSA in 8 millilitersPBS, for 30 minutes at 37° C. The SPDP-HSA is then activated by additionof 125 micrograms DTT, which results in the formation of a thiolactivated protein. Both the mAb-LC-SMCC and the thiol activated HSA arethen dialyzed to PBS at 4° C. using a membrane with a 10-14 kDa cutoffto remove residual unreacted LC-SMMC, SPDP and DTT. The mAb-LC-SMCC andthe thiol activated HSA are then mixed together and incubated at 37° C.for 30 minutes to allow the thiol group on HSA to react with themaleimide end of the LC-SMCC on the mAb, resulting in the formation of acovalent thio-ether bond. After the coupling reaction, the mixture isdialyzed again towards PBS using a membrane with a 100 kDa cut off, toremove any unreacted HSA from the mixture.

A similar reaction is performed to generate a control conjugateincluding human serum albumin (HSA) conjugated to a non-sialoadhesinbinding antibody, mAb 13D12.

Samples taken in between different steps of the cross-linking protocolmay be analyzed to confirm formation of a conjugate. For example, suchsamples may be separated by SDS-PAGE on a 7% gel and proteins stainedwith a reagent such as Coomassie blue in order to visualize thereactants and reaction products.

Example 7

Internalization of a conjugate composition including a sialoadhesinbinding moiety and a cargo moiety is demonstrated in primarymacrophages. In this example, the HSA-mAb 41D3 conjugate and HSA-mAb13D12 conjugate are incubated for 1 hour at 37° C. with sialoadhesinexpressing primary porcine macrophages. Cells in separate culture dishesare incubated for 1 hour at 37° C. with mAb 41D3, mAb 13D12 or with HSAalone. Cells are then washed, fixed by incubating with 3%paraformaldehyde for 10 minutes and permeabilized by incubating with0.1% Triton X-100 for 2 minutes.

HSA is detected in these preparations by incubating the cells with aHSA-specific biotinylated polyclonal pig serum, followed by incubationwith FITC-labeled streptavidin FITC (Molecular Probes). The monoclonalantibodies are detected with TxRed-labeled goat-anti-mouse Ig (MolecularProbes). The cells are then analyzed using an appropriate technique,such as confocal microscopy.

Confocal analysis is performed using a scanning spectral confocalsystem, such as a Leica TCS SP2 laser linked to a Leica DM IRBE invertedmicroscope, from Leica Microsystems GmbH. Image acquisition is performedusing a Leica TCS SP2 confocal software package and overlay images areproduced with Adobe Photoshop CS.

Analysis demonstrates mAb 41D3 internalization both when it is coupledto HSA or not, indicating that the coupling reaction had no effect onthe ability of mAb 41D3 to bind to sialoadhesin and to induceinternalization. Internalization of free HSA is either absent, or atvery low levels when it is added to macrophages not coupled to mAb 41D3,but a clear internalization of HSA is observed when it is coupled to mAb41D3. Further, internalized HSA co-localizes with mAb 41D3 in confocalimages of cells treated with the HSA-mAb 41D3 conjugate. Coupling HSA tomAb 41D3 results thus in co-internalization of HSA with mAb 41D3 via thesialoadhesin receptor.

Thus, contact of a conjugate including HSA coupled to thesialoadhesin-specific mAb 41D3 with primary, sialoadhesin expressingmacrophages, results in sialoadhesin-dependent uptake of HSA intomacrophages, while addition of non-coupled HSA to macrophages did notresult in efficient HSA uptake.

Example 8

Immunization is performed using conjugate compositions herein in thisexample. Six week old conventional pigs are purchased from a porcinearterivirus negative farm and housed in isolation units with HEPAfiltered air following the recommendations of the ethical committee ofthe Faculty of Veterinary Medicine, Ghent University. Six pigs areimmunized with one milligram of a conjugate having HSA coupled to thesialoadhesin-specific mAb 41D3. Three pigs are immunized with onemilligram of a control conjugate having HSA coupled to the control mAb13D12. Each immunization includes administration of the conjugate in 3milliliters PBS, of which 1.5 milliliters is administered intravenouslyand 1.5 milliliters is administered intramuscularly. As a control, sixpigs are immunized with one milligram unconjugated HSA.

Blood samples are collected before immunization and at days 10, 17, 24,32 and 38 after immunization. Three months later, blood is sampled againand the pigs are boostered with one milligram HSA by intramuscularinjection.

Serum obtained from immunized pigs is analyzed for the presence ofHSA-specific IgM and IgG antibodies by ELISA. The HSA-specific IgM, andIgG antibody titers are determined with an indirect ELISA as describedin Y. E. Van der Stede et al., 2001, Vaccine 19:1870-8; and F. Verdoncket al., 2005, J. Control Release 104:243-58. Briefly, the wells of a96-well Polysorb Immuno microtiter plate (NUNC) are coated with HSA at aconcentration of 30 micrograms/milliliter in PBS for 2 hours at 37° C.The plates are then washed and the remaining binding sites are blockedovernight at 4° C. with PBS supplemented with 0.2% TWEEN®80. Two-foldserial dilutions of the serum samples (starting from 1/10) in ELISAdilution buffer (PBS+0.05% TWEEN®20) are added to the plate, followed bythe swine-specific IgM, or IgG MAb, such as described in D. Van Zaaneand M. M. Hulst, 1987, Vet Immunol. Immunopathol. 16:23-36, andperoxidase-conjugated rabbit-anti-mouse polyclonal antibodies (Dako)supplemented with 2% pig serum. ABTS and H₂O₂ are used as chromogen andsubstrate and the optical density is spectrophotometrically measured at405 nm (OD405). The cut-off values are calculated as the meanOD₄₀₅-value of all sera (dilution 1/10) at day 0, increased with threetimes the standard deviation. The antibody titer is the inverse of thehighest dilution that still had an OD₄₀₅ higher than the calculatedcut-off value.

FIG. 2 shows means of HSA specific IgM (FIG. 2A) and IgG (FIG. 2B) serumtiters (±SEM) after primary immunization. FIG. 2C shows means of HSAspecific IgG serum titers after booster immunization. Square symbolsindicate pigs immunized with HSA coupled to Sn-specific mAb 41D3;triangle symbols indicate pigs immunized with HSA coupled to irrelevantcontrol mAb; and circle symbols indicate pigs immunized with free HSA.

After primary immunization, low to undetectable titers of IgM antibodiesare detected in the pigs immunized with HSA alone, or with HSA coupledto the control mAb 13D12. In contrast, IgM antibodies are presentstarting from 10 days post immunization (dpi) in the pigs immunized withHSA coupled to the sialoadhesin-specific mAb 41D3. These antibodiesremained at a nearly constant level until 17 dpi, and started to declinefrom 24 dpi as illustrated in FIG. 2A.

Similarly, HSA-specific IgG antibodies are undetectable in pigsimmunized with HSA alone until 24 dpi, and low titers are detected from32 dpi. In pigs immunized with HSA coupled to the control mAb 13D12, lowtiters of HSA-specific IgG antibodies could be detected from 10 dpiwhich reached maximum titers at 32 dpi. Cross-linking of HSA and anon-sialoadhesin binding antibody stimulates some HSA-specific IgGantibody response. In contrast, pigs immunized with HSA coupled to thesialoadhesin-specific mAb developed high titers of IgG antibodiesalready starting at 10 dpi. Maximum antibody titers are detected at 17dpi and these remained constant until 38 dpi as illustrated in FIG. 2B.

To investigate if immunization with HSA coupled to thesialoadhesin-specific mAb 41D3 had an effect on the induction ofHSA-specific memory cells, all animals are boosted 3 months afterprimary immunization with HSA alone. At the time of the boosterimmunization and at 4 dpi, all animals had low to undetectableHSA-specific IgG titers. Starting from 7 dpi, an IgG antibody responseis detected in all animals, but the highest titers are detected in theanimals which received HSA coupled to the sialoadhesin-specific mAb 41D3as the primary immunization as is shown in FIG. 2C.

Pigs immunized with the HSA-mAb41D3 constructs showed the highest IgGand IgM antibody titers throughout the study, which indicates thatcoupling HSA to the sialoadhesin-specific mAb greatly enhances both thespeed of induction and the titers of HSA-specific IgG and IgMantibodies.

Thus, targeted delivery of an immunogen to macrophages is possible bycoupling the immunogen to the sialoadhesin-specific mAb, and thisaffects the humoral immune response, enhancing both the speed ofinduction and the titers of antigen-specific antibodies.

Example 9

Sialoadhesin binding moiety/viral protein conjugate, administration andimmune response. In this example, influenza virus hemagglutinin (HA) isconjugated to sialoadhesin binding moiety monoclonal antibody (mAb)41D3. Influenza virus hemagglutinin conjugated to sialoadhesin bindingmoiety monoclonal antibody 41D3 is either the native protein purifiedfrom virus or a recombinant form produced in eukaryotic cells.Hemagglutinin conjugated to sialoadhesin binding moiety monoclonalantibody 41D3 is chemically cross-linked with mAb 41D3 in this exampleand injected in pigs to demonstrate and evaluate the capacity of theconjugates to induce HA-specific antibodies.

Purification of Native Hemagglutinin

In order to obtain native hemagglutinin, a split H1N1 component isprepared essentially as described by Van Reeth et al., Vet. Rec., 2003.153(1): p. 9-13. Ten-day-old embryonated SPF chicken eggs are inoculatedwith the H1N1 swine influenza strain A/swine/Belgium/1/98. Allantoicfluid is collected 72 hours post inoculation and red blood cells andcell debris are removed via centrifugation. The clarified allantoicfluid is then centrifuged to pellet the virus, 70,000 g at 4° C. for 90minutes. Virus pellets are resuspended overnight at 4° C. in TSE buffer,10 mM Tris-HCl pH7.4, 100 mM NaCl and 1 mM EDTA. Presence of influenzavirus is confirmed with a hemagglutination (HA) test such as describedby K. S. Van Reeth et al., Vet. Rec., 2003. 153(1): p. 9-13 followed byconcentration and purification via ultracentrifugation on a linear 20 to60% (w/v) sucrose gradient, 130,000 g at 4° C. for 14 hours. Gradientfractions containing virus are identified with an HA test, pooled,dialyzed in a slide-a-lyzer dialysis cassette, 10,000 MWCO, againstphosphate buffered saline (PBS) to remove sucrose and concentrated bydialysis in a 20% polyethylene glycol (PEG-20,000) solution. Finally,the hemagglutinin is released from the purified and concentrated virusby centrifugation on a linear denaturing 20 to 60% (w/v) sucrosegradient consisting of 0.1% TWEEN® 80 and 1.2% sodium deoxycholate inTSE buffer, 130,000 g at 4° C. for 14 hours. Fractions containinghemagglutinin are identified with an HA test, pooled, dialyzed in aslide-a-lyzer cassette (10,000 MWCO) against PBS and concentrated bydialysis in a 20% PEG solution. Residual infectious virus is inactivatedby UV treatment of the solution (5 J/cm²). Complete inactivation isconfirmed by inoculation on MDCK cells and two blind passages in10-day-old embryonated SPF chicken eggs.

The purification process is analyzed via SDS-PAGE followed by Westernblotting and Coomassie blue staining. HA is clearly present in theoriginal allantoic fluid, but also in the purified solution after thedenaturing sucrose gradient. HA can be detected as a monomer and as twodifferent multimers, most likely a dimer and a trimer during all stepsof the purification process. FIG. 6A shows SDS-PAGE analysis of thepresence and purity of native influenzavirus hemagglutinin in differentfractions obtained during purification includes detection of HA viawestern blotting using a monoclonal antibody directed against HA of theH1N1 virus. FIG. 6B shows detection of all proteins in the samples isaccomplished via Coomassie blue staining. In both FIGS. 6A and 6B: LaneA: marker, lane B: allantoic fluid after removal of RBC, lane C:allantoic fluid after removal of cell debris, lane D: supernatant afterpelleting the virus, lane E: the virus pellet (1/100 dilution), lane F:virus after the first sucrose gradient and after removal of sucrose(1/100 dilution), lanes G-J: virus after denaturing sucrose gradient:lane G: fraction with HAU 64 and 128 (1/100), lane H: fraction withHAU≧256 (1/100), lane I: fraction with HAU 64 and 128 (undiluted), laneJ: fraction with HAU≧256 (undiluted). HA can be detected as a monomerand as two different multimers, most likely a dimer and a trimer.

Production of Recombinant Hemagglutinin

In further embodiments, a recombinant influenza virus hemagglutininprotein is produced. The recombinant influenza virus hemagglutininprotein used in this example includes the extracellular domain ofhemagglutinin fused to the V5-His tag in the pcDNA3.1 D/V5-His vector(Invitrogen). Viral RNA is isolated from H1N1 swine influenza strainA/swine/Belgium/1/98 via the RNEASY® mini kit (Qiagen) and subsequentlyconverted into cDNA via random primers (Invitrogen) and SuperScript IIreverse transcriptase (Invitrogen) followed by an RNase H (Gibco)treatment. The obtained single stranded cDNA serves as template for PCRamplification of the HA sequence using following primers: forward primerSEQ ID NO:1 and reverse primer SEQ ID NO:2 (Invitrogen). The PCRfragment is then cloned in the pcDNA3.1D/V5-His vector. The sequence isverified via restriction digest and sequencing. The isolated andverified nucleotide sequence encoding the extracellular domain ofinfluenza virus hemagglutinin is shown and referred to as SEQ ID NO:3herein.

Extracellular Domain of Influenza Virus Hemagglutinin—SEQ ID NO:3

Production and purification of the recombinant, soluble HA isdemonstrated in a human embryonic kidney cell line, HEK293T. HEK293Tcells are transfected using calcium phosphate to produce the solublehemagglutinin. Sixteen hours post-transfection, medium is replaced byfresh medium with or without fetal bovine serum (FBS). Samples are takenevery 24 hours post transfection and analyzed via SDS-PAGE and westernblotting to determine at what time post transfection the supernatantcontains the highest concentrations of soluble HA (FIGS. 7A and 7B). Therecombinant, soluble HA is produced in HEK293T cells, no matter whetherFBS is present in the serum or not. In the absence of FBS, the maximumamount of HA in the serum is reached at 72 hours post transfection. Inthe presence of FBS, the amount of HA stays the same until 120 hourspost transfection. The recombinant HA is produced as a monomer and, to alesser extent as a trimer, which is confirmed by the disulfide-reducingagent beta-mercaptoethanol.

FIGS. 7A and 7B show SDS-PAGE analysis of the production of recombinantHA with a V5-His tag. The recombinant HA is produced in the absence,FIG. 7A or in the presence FIG. 7B of fetal bovine serum. Samples aretaken every 24 hours post transfection as indicated above the lanes inFIGS. 7A and 7B. HA is detected via a monoclonal antibody recognizingthe V5 tag. Under non-reducing conditions, HA is mainly present in thesupernatant as a monomer, although it also forms trimers. In thepresence of the disulfide-reducing agent beta-mercaptoethanol, indicatedwith an asterisk* in FIGS. 7A and 7B, HA is only present as a monomer,confirming that the high molecular weight protein was indeed an HAtrimer. The molecular weight of the proteins is determined via a markerin lane A (prestained) and B.

After collection of the supernatant, the recombinant HA is purified viaNi-NTA beads according to the manufacturer's instructions (Qiagen).Because of interference of the FBS with this purification step, HA isfurther produced without FBS and the supernatant is collected at 72hours post transfection. Different fractions are taken during thepurification process and HA is visualized via SDS-PAGE followed byWestern blot or Coomassie blue staining as shown in FIGS. 8A and 8B,respectively. The recombinant HA is present in the original supernatant,but not in the flow through. HA is clearly concentrated, both themonomer and the trimer. FIGS. 8A and 8B show SDS-PAGE analysis of thepurification process of recombinant HA-V5-His via Ni-NTA beads. SDS-PAGEis followed by Western blotting and detection of HA via a monoclonalantibody directed against the V5-tag to identify the fractionscontaining HA, FIG. 8A, or by Coomassie blue staining to visualize thepurity of the HA, FIG. 8B. Lane A: marker, lane B: original supernatantwith FBS, lane C and D: original supernatant from two differentproductions without FBS, lane E: flow through of purification, followinglanes: elution fractions of 0.8 ml, fractions are indicated with theirrespective number above the lanes. HA is present in all originalsupernatants but not in the flow through. HA is clearly concentrated,both the monomer and the trimer.

Conjugation of Antibodies with HA

Hybridomas producing monoclonal antibody 41D3, described in X. Duan etal., Adv. Exp. Med. Biol., 1998. 440: p. 81-8, or monoclonal antibody13D12, described in H. J. Nauwynck and M. B. Pensaert, Arch. Virol.,1995. 140(6): p. 1137-46, directed against porcine sialoadhesin or anisotype matched (IgG1) irrelevant control antibody, respectively, arecultivated and supernatant is collected every 72 hours. Antibodies arepurified via protein G sepharose columns as described by themanufacturer (GE Healthcare).

Influenza virus hemagglutinin Type A/swine/Belgium/1/98 having proteinsequence identified as GenPept Accession number AY590824, and herein asSEQ ID NO:4, is used in this example as a cargo moiety conjugated to mAb41D3.

The purified antibodies are coupled to influenza hemagglutinin (HA) SEQID NO:4 via a disulfide-bridge. To accomplish this, the 41D3 monoclonalantibody, the isotype matched control monoclonal antibody and HA areactivated with the cross-linker SPDP(N-succinimidyl-3-(2-pyridyldithio)-propionate) according to themanufacturers' instructions (Pierce Biotechnology). For HA, the SPDP isactivated via dithiothreitol (DTT). The activated proteins are purifiedfrom the unreacted cross-linkers via PD-10 desalting columns (AmershamBiosciences). The activated proteins are mixed in a 1:1 antibody:HAratio. The uncoupled HA is removed from the coupled products, 41D3-HAand control monoclonal antibody-HA, by dialysis with a float-a-lyzer(Spectra/Por) with a MWCO 100,000.

Coupling of the antibodies and HA to form conjugates is verified viaSDS-PAGE followed by western blotting and by analysis of uptake of thecoupling products by primary alveolar macrophages. For both antibodiesthere is a clear shift towards a bigger protein, which confirms thateach antibody is coupled with HA. FIGS. 9A and 9B show visualization ofcoupling of antibodies 13D12, FIG. 9A, or 41D3, FIG. 9B, with isolatednative HA. Samples taken during the coupling process are analyzed viaSDS-PAGE followed by western blotting and detection via a mixture of 3monoclonal antibodies recognizing HA of H1N1. Lane A: original antibody,lane B: SPDP treated antibody after PD-10 desalting column, lane C: HAcoupled with antibody, lane D: HA coupled with antibody after dialysisand lane E: marker.

Vaccinations

Twelve (12) six-week-old pigs are obtained from an influenzavirus-seronegative farm and randomly assigned to three groups of fourpigs. The animals are housed in isolation units with high efficiencyparticulate air (HEPA) filters. Water and feed are provided ad libitum.The first group of four pigs is immunized with 1 mg HA-13D12 conjugateper pig, the second group with 1 mg HA-41D3 conjugate per pig, and thecontrol group with the same volume of PBS without any protein. For eachpig, the conjugate is diluted in 3 ml PBS. Half of the conjugate isinjected intravenously and the other half intramuscularly in the neck.

Blood samples are collected from all pigs at the time of immunizationand on day 4, 7, 11, 14 and 18 after immunization. The sera are examinedin hemagglutination inhibition (HI) tests, virus neutralization (VN)test and in immunoperoxidase monolayer assays (IPMA) as described in K.Van Reeth, S. Van Gucht, and M. Pensaert, Vet. Rec., 2003. 153(1): p.9-13.

Hemagglutination Assay (HA)

Samples containing influenzavirus hemagglutinin are serially diluted andmixed with 0.5% chicken erythrocytes for one hour at room temperature.The highest dilution of that still shows hemagglutination is consideredto be the hemagglutinating titer.

Hemagglutination Inhibition (HI)

The sera are examined in a hemagglutination inhibition (HI) test againstH1N1 strain A/swine/Belgium/1/98. The inactivated sera are first treatedwith receptor-destroying enzyme (RDE) from Vibrio cholera, followed byinactivation of the enzyme via sodium citrate treatment. Afterwards, thesera are absorbed on chicken erythrocytes to remove non-specificinhibitors of influenza hemagglutination. The HI test is carried outaccording to standard procedures including positive and negativecontrols. Because of the pretreatments, the starting dilution of thesera was 1:10 followed by two-fold serum dilutions. Furthermore, eachwell was mixed with four hemagglutination units of the H1N1 strain and0.5% chicken erythrocytes. After 1 hour incubation, the results areinterpreted. In the presence of HA recognizing antibodies, nohemagglutination can be observed and the RBC will all be together in onespot on the bottom of the plate. The HI titer is the reverse of thetiter needed for complete inhibition of hemagglutination. As areference, positive and negative control sera are included in the HItests.

Virus Neutralization (VN)

Sera are also examined in a virus neutralization (VN) test for thepresence of H1N1 neutralizing antibodies. Two-fold serum dilutions areincubated with 100 tissue culture infectious doses (TCID₅₀) ofA/swine/Belgium/1/98 virus. Madin-Darby canine kidney (MDCK) cells arethen added at a concentration of 600,000 cells per ml. After 24 hoursincubation, virus-positive cells are detected by immuno-peroxidasestaining. Starting dilution of the sera was 1:2.

Immuno-Peroxidase Monolayer Assay (IPMA)

Finally, sera are analyzed via an immuno-peroxidase monolayer assay(IPMA) for the presence of influenza recognizing antibodies. Therefore,MDCK cells are grown for 24 hours in the presence of 1000 TCID₅₀ ofA/swine/Belgium/1/98 virus. After fixation, cells were incubated withtwo-fold serum dilutions followed by immuno-peroxidase staining forantibody detection. Starting dilution of the sera is 1:2. Results areshown in FIG. 12. FIG. 12 shows mean immuno-peroxidase monolayer assay(IPMA) titers of pigs immunized with 13D12-HA or 41D3-HA. For eachgroup, four pigs are immunized with native hemagglutinin (HA) coupledwith the isotype matched (IgG1) control antibody 13D12, or HA coupledwith 41D3, a monoclonal antibody directed against porcine sialoadhesin.Serum is collected at day 0, 4, 7, 11, 14 and 18 after immunization andanalyzed via an IPMA. Pigs immunized with HA coupled to the sialoadhesinspecific mAb 41D3 show a faster induction and higher titers of IPMAantibodies.

Example 10

A conjugate composition including a sialoadhesin binding moiety and acytotoxic agent is generated in this example. The cytotoxic agentsaporin is a 30 kDa plant enzyme, belonging to the family of ribosomeinactivating proteins (RIP). Saporin may be isolated from seeds of theplant Saponaria officinalis according to methods known in the art orobtained commercially. Saporin is used in this example as arepresentative of cytotoxic agents which may be included in a conjugateherein.

Saporin alone has no cell binding moiety and can thus not enter thecell. However, following conjugation with a sialoadhesin binding moietyto produce a conjugate, saporin is co-internalized in the cell alongwith the sialoadhesin binding moiety. Saporin is also representative ofcytotoxic agents which are capable of linkage to a sialoadhesin bindingmoiety by a disulfide bond between the sialoadhesin binding moiety andsaporin. Further cytotoxic agents are described in G. R. Thrush et al.,Annu. Rev. Immunol., 1996. 14: p. 49-71. The disulfide bond between thesialoadhesin binding moiety and saporin allows dissociation of toxin.

Conjugation of a Cytotoxic Agent with a Sialoadhesin Binding Moiety

The purified antibody 41D3 is conjugated to saporin (Sigma) via adisulfide-bridge. Therefore, the antibody and saporin are activated withthe cross-linker SPDP (N-succinimidyl-3-(2-pyridyldithio)-propionate)according to the manufacturers' instructions (Pierce Biotechnology). Forsaporin, the SPDP is activated with dithiothreitol and the proteins arepurified from the unreacted cross-linkers with PD-10 desalting columns(Amersham Biosciences). The activated proteins are mixed in a 1:1antibody:saporin ratio. The uncoupled saporin was removed from thecoupling products by dialysis with a float-a-lyzer (Spectra/Por) with aMWCO 100,000.

Coupling of saporin and antibody 41D3 is verified via SDS-PAGE followedby Coomassie blue staining and by analysis of uptake of the couplingproducts by primary alveolar macrophages. Coomassie blue staining of anSDS-PAGE shows a clear, upwards shift after conjugation, indicating anincreased size, which confirms that part of the antibodies are coupledwith saporin. The uncoupled proteins are clearly removed after dialysis.FIG. 11 shows SDS-PAGE and Coomassie blue staining of different samplestaken during the antibody-saporin conjugation protocol. Lane I: marker,lane A: original antibody, lane B: SPDP treated antibody, lane C: SPDPtreated antibody after PD-10 desalting column, lane D: original saporin,lane E: SPDP and DTT treated saporin, lane F: SPDP and DTT treatedsaporin after PD-10 desalting column, lane G: saporin coupled withantibody, lane H: saporin coupled with antibody after dialysis, andfinally lane J: saporin coupled with antibody in the presence of thedisulfide-reducing agent beta-mercapto-ethanol in the loading dye. Forboth antibodies there is a clear shift towards a bigger protein, whichconfirms that part of the antibodies are coupled with saporin. Theuncoupled proteins are clearly removed after dialysis.

Saporin is conjugated to the mAb 41D3, resulting in a conjugatecomposition herein. A control conjugate including a non-sialoadhesinrecognizing antibody, mAb 13D12 and saporin is also generated.

The mAb 41D3/saporin conjugate and the control mAb 13D12/saporinconjugate are each separately incubated with sialoadhesin expressingprimary porcine macrophages. After an appropriate time, the cells areimmunostained and analyzed by confocal microscopy to determine whetherthe conjugate binds and is internalized into the cells. Incubation ofcells with the mAb 41D3/saporin conjugate results in internalization ofthe conjugate, indicating that the 41D3 mAb is still functional to bindand stimulate internalization of the conjugate. In contrast, the mAb41D3/saporin conjugate is observed not to be internalized.

Example 11

Cytotoxic effects of the mAb 41D3/saporin conjugate and the mAb13D12/saporin conjugate on primary porcine macrophages are tested.Macrophages are incubated with various concentrations of either the mAb41D3/saporin conjugate or the mAb 13D12/saporin conjugate for variousperiods of time. An MTT assay is used to colorimetrically assay cellpopulations and differentiate living and dead cells.

The following table shows OD values as a function of time and thepercentage of living cells as a function of conjugate concentration as aresult of these treatments:

Macrophages

OD values: 0 10 30 100 APOP Levend 41D3-Sap 0.772 0.537 0.541 0.4012.644 13D12-Sap 0.851 0.708 0.703 0.780 0.445 2.824 Average 0.389 0.423

% Living cells 0 10⁻⁷ 0.3*10⁻⁶ 10⁻⁶ 41D3-Sap 89.8 29.3 30.4 13D12-Sap110.2 73.4 72.1 91.9

FIG. 3 depicts the percentage of living cells as a function of conjugateconcentration in graphical form.

Example 12

A mAb 41D3/saporin conjugate is incubated with various cells to assessthe effect of the cytotoxic agent saporin. CHO cells that expressrecombinant sialoadhesin and CHO cells do not express sialoadhesin areeach incubated with various amounts of the mAb 41D3/saporin conjugateand effects on cell viability are measured at various times followingaddition of the conjugate. An MTT assay is used to colorimetricallyassay cell populations and differentiate living and dead cells.

The following table shows OD values as a function of time and thepercentage of living cells as a function of conjugate concentration as aresult of these treatments:

CHO (41D3-Sap)

OD values: 0 6.25 12.5 25 50 CHO-K1 3.475 3.379 3.299 2.873 2.822 CHO-Sn2.054 1.45 1.474 1.354 1.47

% Living cells 0 10⁻⁷ 0.1*10⁻⁶ 0.25*10⁻⁶ 0.5*10⁻⁶ CHO-K1 100.0 96.9 94.280.3 78.6 CHO-Sn 100.0 63.0 64.4 57.1 64.2

FIG. 4 depicts the percentage of living cells as a function of conjugateconcentration in graphical form. CHO-Sn indicates CHO cells expressingsialoadhesin. CHO-K1 indicates CHO cells which do not expresssialoadhesin.

In Vivo Treatment of Pigs with Saporin-41D3 Immunotoxin

Pigs are injected intramuscularly with 0.1 or 1 mg saporin-41D3conjugate in 1 ml of PBS/kg body weight, either as a single dose, ordivided in two doses injected with an interval of 6 hours. Four pigs areused for each saporin-41D3 conjugate condition, four control pigsinjected with PBS alone; twenty pigs in total. The pigs are euthanized24 hours after the first injection and the local, draining lymph nodesare collected analyzed.

Flow Cytometry Analysis of Lymph Node Immune Cells

Changes in the immune cell population of the lymph nodes are analyzed byflow cytometry. Total immune cells are prepared from lymph nodes bymechanical dissociation or collagenase D digestion. For mechanicaldissociation, lymph node samples are dissociated with needles andfiltered on a 40 micron pore size nylon filter. Cells are collected intoa 50-ml conical tube and washed twice in RPMI. For collagenase Ddigestion, lymph nodes are incubated for 1 hour at 37° C. in RPMI with 1mg collagenase D (Sigma)/ml. Cell suspensions are then filtered througha 40 micron pore size nylon filter and collected in RPMI with 30% FBS.After centrifugation for 15 minutes at 400×g, cells are washed threetimes with RPMI with 5% FBS. For erythrocyte lysis, cells are incubatedfor 5 minutes in 5 ml lysis solution (NH4Cl [0.15 M], KHC03 [1 mM],Na²⁺EDTA [0.1 mM]) and washed three times in RPMI with 5% FBS.Monocyte/macrophage cells are identified with FITC-labeled SWC3 specificmAb 74-22-15. Sialoadhesin expressing macrophages are stained withbiotinylated mAb 41D3, followed by FITC labeled streptavidin. TotalT-cells are quantified by staining with a FITC-labeled CD3-specific mAb,while subpopulations of T-cells are quantified by staining with eitherFITC-labeled CD4 or CD8. B cells are identified with a FITC-labeledmouse monoclonal anti-pig IgM antibody (Clone M160).

Analysis of Lymph Node Micro-Anatomy

Samples of lymph nodes are fixed in a phosphate-buffered 3.5%formaldehyde solution for 24 hours. After fixation, the samples areembedded in paraffin using an automated system (Shandon Citadel TissueProcessor, Cheshire, UK). Sections of 8 microns in thickness are made,deparaffinized in xylene, rehydrated in descending grades of alcohol,stained, dehydrated in ascending grades of alcohol and xylene, andmounted on slides with DPX. Hematoxylin-eosin staining is done toanalyze the morphology and micro-anatomy of the lymph nodes of treatedand untreated pigs.

Immunohistochemical Analysis

Samples from the draining lymph nodes are embedded in methylcellulosemedium and frozen at −70° C. Cryostat sections (5 to 8 microns inthickness) are made and fixed in acetone for 20 minutes at −20° C.Sections are stained with one or more of the following:

-   -   (1) FITC-labeled goat-anti-mouse IgG antibodies to detect the        injected immunotoxin;    -   (2) biotinylated mAb 41D3 followed by FITC-labeled streptavidin        to allow quantification of sialoadhesin expressing cells and        evaluate the effect of the immunotoxin on the numbers of        sialoadhesin expressing cells;    -   (3) biotinylated antibody 74-22-15, an anti-SWC3 antibody which        stains all monocytes and macrophages, not only the sialoadhesin        expressing cells, followed by FITC-labeled streptavidin to        assess the effect of the toxin on all cells of the monocyte        macrophage lineage; and    -   (4) biotinylated mAb 41D3 followed by FITC-labeled streptavidin        together with a staining with a goat polyclonal antibody        specific for activated caspase-3 (apoptosis marker) followed by        TexasRed-labeled rabbit-anti-goat antibodies to quantify the        total level of apoptosis and the number of apoptotic        sialoadhesin expressing macrophages in the draining local lymph        nodes of saporin-41D3 conjugate immunotoxin treated pigs.

In vivo administration of a sialoadhesin binding moiety/cytotoxic agentconjugate, e.g., a saporin-41D3 conjugate allows for assessment of thein vivo functionality of a sialoadhesin specific immunotoxin andassessment of the capacity of the conjugate to selectively killsialoadhesin expressing macrophages in lymph nodes. Both in vitro and invivo assays provide information on the dose and manner of administrationthat is optimal for depletion of sialoadhesin expressing macrophages inlymph nodes. Depletion of sialoadhesin expressing macrophages may haveutility in treatment of specific diseases that involve macrophages,e.g., rheumatoid arthritis, inflammatory skin diseases, persistentinfections and others.

Example 13 Patients

Synovial membrane biopsies were obtained by needle arthroscopy of eightpatients with CCP positive undifferentiated arthritis (UA CCP), 21patients with early rheumatoid arthritis (ERA) and 13 patients withmethotrexate resistant RA (Mtx res RA). Synovial membrane biopsies of 15non-diseased (control) patients were used as controls. This study wasapproved by the local ethical committees and informed consents weresigned by the patients.

Anti-Sn Antibody Production and Coupling to Methotrexate

Monoclonal Abs were generated in rat by immunization with a solubleextracellular part (AA 20-1339) of mouse Sn (Synaptic Systems, Germany).Lymph nodes were isolated and fused with myeloma cells to generate ahybridoma cell line. Clones were tested for their ability to bind thesoluble mSn and mSn expressed on CHO cells and subsequently subclonedtwice to guarantee their monoclonal nature. The hybridoma for theisotype control was purchased at DSHB, Iowa and treated similar to therat anti-mSn Ab.

Hybridomas were grown to confluency in DMEM 10% IgG depleted FCS, 1% P/Sand medium was harvested up to three times.

The Ab was purified from the medium on a protein G column, proteincontaining fractions were pooled and either dialyzed to PBS or directlycoupled with MTX.

Activated MTX (in dimethylformamide) was coupled to the rat anti-mSn Abin a 20 to 1 ratio. Coupling of MTX to the rat anti-mSn Ab is done by anactive ester intermediate. MTX (45 mg/ml), N-hydrosuccinimide (NHS) (24mg/ml) and dicyclohexylcarbodiimide (DCC) (42 mg/ml) are dissolved indimethylformamide (DMF). 1 ml of MTX is mixed with 0.5 ml of NHS and 0.5ml of DCC. The solution is rotated in the dark at room temperature for 1hour and subsequently at 4° C. for 18 hours. During this step andactivated ester is formed, which will form a stable covalent amide bondwith the Lys residues in the rat anti-mSn Ab. After activation of MTXthe precipitate is removed by centrifugation and the liquid is stored at−20° C. in a dry environment. Activated MTX is mixed with Ab in a20-fold molar excess and rotated at 4° C. for 4 hours. Uncoupled MTX isremoved by gel permeation. Protein concentrations were measured at 280nm, the MTX content at 370 nm.

CIA Induction, Treatments and Evaluation in Mice

Eight-week-old DBA/1 Rj (H-2q background) mice were obtained fromJanvier, France. Mice were immunized intradermally at the base of thetail with 200 μg of chicken type II collagen (CII) (Morwell DiagnosticsGmbH, Zurich, Switzerland) (in 0.1 M acetic acid) emulsified inIncomplete Freund's Adjuvant+mycobacterium Tuberculosis H37RA (150μg/mouse) (Difco, Lawrence, Kans., USA). Twenty-one days later, micewere re-challenged with an injection of CII in Incomplete Freund'sAdjuvant.

Mice were treated intraperitoneally twice a week from day 14 afterinduction on until sacrifice. Twelve mice were treated per condition.Treatments consisted out of PBS as a negative control, a high dose ofMTX (Sigma) (35 mg/kg=700 μg/mouse), isotype antibody conjugated withMTX (iso-MTX; 200 μg/mouse, equivalent with 4 μg/mouse MTX or 0.2 mg/kg)and the anti-Sn antibody conjugated with MTX (ab-MTX; 200 μg/mouse,equivalent with 4 μg/mouse MTX or 0.2 mg/kg) in PBS.

From Day 21, mice were monitored for clinical symptoms of arthritisuntil the day of sacrifice (Day 42). Clinical severity was graded asfollows: 0=normal; 0.5=erythema and edema in only one digit; 1=erythemaand mild edema of the footpad, or ankle or two to five digits;2=erythema and moderate edema of two joints (footpad, ankle, two to fivedigits); 3=erythema and severe edema of the entire paw; 4=reducedswelling and deformation leading to incapacitated limb. The individualmouse arthritic score was obtained by summing the scores recorded foreach limb. Clinical evaluations were performed by two investigatorsunaware of mouse identity and the mean of both scores was calculated.

ELISA

The serum concentrations of Ab-MTX after injection were measured in anELISA setup. The rat Ab was captured from solution by an anti-rat Ab(goat anti-rat-AF488 A11006, Invitrogen) and detected with an HRPlabeled anti-rat Ab (goat anti-rat-HRP 112-035-167, JacksonImmunoResearch). Concentrations were presented as concentrations withthe concentration 5 minute after injected set as 100%.

Isolation of Alveolar Mouse Macrophages and Detection of Internalized Sn

Alveolar macrophages were isolated from BALB/c mice by lavage with 1%versene in PBS. CHO or macrophages were coated on glass coverslips usingpoly-L-lysine for 1 hour. Next the cells are incubated with ratanti-mSn, rat anti-mSn-MTX and isotype controls at 1 μg/ml both at 4° C.and 37° C. After 90 minutes the cells were fixed with 4%paraformaldehyde and permeabilized with 0.5% saponin. Surface bound orinternalized rat anti-mSn was detected with an Alexa Fluor 488 labeledgoat anti-rat Ab.

Immunohistochemistry and Immunofluorescence on Human Synovial Tissue

IHC and IF were performed on frozen sections of synovial biopts afterfixation in aceton. The primary mouse anti-human Sn antibody (clone 7D2;Santa Cruz) was detected using the universal LSAB kit (Dako) containingan anti-mouse biotinylated antibody and streptavidin-HRP. Chromogendeposition was obtained by the use of AEC substrate (Dako). The level ofexpression was independently scored by two observers. Asemi-quantitative four-point scale was used with zero representing thelowest and three representing the highest level of expression. Thescoring was calibrated for synovial lining and sublining tissueseparately by examining a representative number of samples. Each sectionwas scored twice per observer and absolute intraclass correlationcoefficients were all above 0.85. Multiple comparisons were performedfor Sn expression in the lining and sublining synovial layer between thefour groups.

For colocalization of Sn, CD209 and CD68, aceton fixed frozen biopts ofthree patients per group were blocked with 2% goat and 2% donkey serumin 5% BSA in PBS. First mouse anti-CD209 (BD biosciences) antibody wasapplied and detected with Cy3-labeled anti-mouse (Jackson ImmunoResearch). Free antigen-binding moieties were blocked by the addition of10% mouse serum. Mouse IgG1 anti-Sn and biotin-labeled mouse IgG2banti-CD68 (Immunosource) were then added and respectively detected byAlexa Fluor488-labeled anti-mouse IgG1 and Alexa Fluor647-labeledstreptavidin (both Invitrogen). Slides were mounted with Prolonganti-fade mounting medium with DAPI (Invitrogen). Negative controlstainings were performed by using isotype controls or omission of oneprimary antibody. Colocalization was determined by confocal microscopy(Leica TCS LSI).

Immunohistochemistry and Immunofluorescence on Mouse Tissue

IHC was performed on aceton fixed frozen sections of non-decalcifiedmouse knee, obtained by the use of the Cryojane tape transfer system(Leica). The primary rat anti-mouse Sn antibody (clone 3D6.112;AbdSerotec) was detected using containing an anti-rat biotinylatedantibody (eBioscience), streptavidin-HRP from the universal LSAB⁺ kit(Dako) and visualized by the use of AEC substrate (Dako).

For the detection of the injected anti-Sn antibody conjugated to MTX,aceton fixed frozen biopts of spleen or knee were analyzed. Mouse kneeswere decalcified by overnight shaking in 0.5 M EDTA pH 7.5 at 4° C.After 1 hour incubation in 10% sucrose, knees were subsequentlyincubated overnight in 15% sucrose and 15% sucrose/50% tissue teck(Jung) before sectioning. Alexa Fluor546 anti-rat (Invitrogen) wasapplied for detection of the primary injected antibody. Slides weremounted with prolong anti-fade mounting medium with DAPI (Invitrogen)and visualized using wide field microscopy (Leica TCS SP5 II).

Flow Cytometry

Paired peripheral blood—synovial fluid samples were obtained from RA andSpA patients with an active knee synovitis. Patients were diagnosedusing the ACR 2010 criteria for RA or the ASAS 2010 criteria for axialand peripheral SpA. Additionally, healthy control peripheral bloodsamples were collected. The study was approved by the local EthicalCommittee of the Ghent University hospital. PBMC were isolated fromheparinized whole blood by density gradient centrifugation usingHistopaque-1077 (Sigma-Aldrich). Likewise, SFMC were extracted fromsynovial fluid collected in anticoagulant EDTA-coated tubes.

Multi-color flow cytometry was performed using the following antibodies:CD14 V500 (BD; clone M5E2), CD16 PE-Cy7 (eBioscience; clone CB16),HLA-DR APC-eFluor 780 (eBioscience; clone LN3), CD56 V450 (BD; cloneB159); Sn PE (Santa Cruz; clone 7D2); mIgG1 isotype (eBioscience; cloneP3.6.2.8.1). 7-AAD (BD) was used to distinguish alive from dead cells.Samples were acquired on a BD FACS Canto II and analyzed using FlowJosoftware (TreeStar). A geometrical mean was calculated by substractingPE signal from the isotype condition from the PE signal derived from theSn PE antibody.

Statistical Analysis

Absolute intraclass correlation coefficients were calculated for qualityanalysis of the semi-quantitative scoring of Sn IHC staining. Foranalysis of longitudinal clinical scores, mixed model analysis withrandom intercept was used. Differences in clinical (day of onset and day42 score) and histological data between the treatment groups andsemi-quantitative scores of patient groups were assessed by KruskalWallis tests followed by Mann-Whitney-U test with correction using theHolm procedure. Fisher's Exact test was applied to analyze arthritisfrequencies. All analyses were performed using SPSS 19.0 statisticalsoftware (Chicago, Ill., USA).

Increase of Slaloadhesin Expressing Cells Early in Disease andThroughout Rheumatoid Arthritis Disease Progression in Synovial Tissue

Synovial membrane biopsies from non-diseased controls, patients withanti-CCP positive undifferentiated arthritis (UA CCP⁺), early rheumatoidarthritis (ERA) and methotrexate resistant RA (Mtx res RA) were analyzedfor their Sn expression by IHC. Lining and sublining synovial layerswere scored separately. The trend for an increased Sn expression in thelining synovial layer (FIG. 13 a) according to disease severity, is morepronounced and significantly different between groups in the sublininglayer (FIG. 13 b). Sn expression in the sublining is alreadysignificantly increased between the control group and the group with UApatients. Moreover, Sn levels in the sublining are furthermoresignificantly elevated between patients with ERA and patients withmethotrexate resistant RA. To conclude, Sn expression is clearlyup-regulated in early stage, still undifferentiated, arthritis asopposed to a few Sn expressing cells in healthy synovial tissue.Furthermore, aggressive late-stage methotrexate resistant RA displaysclearly more Sn expressing cells than early RA.

Sn expressing cells were also shown to be present in synovial tissue ofpatients with osteoarthritis (OA), spondyloarthritis (SpA), systemiclupus erythematosus (SLE) and psoriatic arthritis (PsA).

Sn is Mainly Expressed on CD209⁺ CD68⁺ Macrophages in Synovial Tissue

Triple immunofluorescence, analyzed by confocal microscopy clearly showscolocalization of Sn expression, mainly on the same cells as CD209(DC-SIGN) and CD68 expression. In addition, some Sn expressing cellswere single positive for CD209 or CD68. These findings were consistentin all groups (control, UA, ERA, MTX resistant RA, SpA), of whichsynovial biopts of three patients each group were stained. To conclude,we have shown that sialoadhesin is mainly expressed on CD209⁺ CD68⁺“Inflammatory MMP producing macrophages” as Van Lent et al. [19] havedescribed in 2003 them to be the macrophages responsible for the matrixtissue degrading MMP1 production.

High Sn Expression on Monocytes, Peripherally in Blood and Locally inSynovial Fluid

Sialoadhesin was found to be highly expressed in blood of controls andpatients on the main monocytic populations, being CD14^(+high) CD16⁻“classical monocytes” and CD14^(+high) CD16⁺ “intermediate monocytes” ascompared to the sialoadhesin negative population of NK cells. Moreover,monocytes in general, but the intermediate monocytes more in particularaccumulate massively in the synovial fluid in the inflamed knee jointsof patients with RA or SpA. Remarkably Sn expression is dramaticallyincreased on the CD14^(+low) CD16⁻ “non-classical monocytes” locally insynovial fluid as compared to peripheral blood of the same patient.

Sn is Highly Abundant in the Inflamed Mouse Knee During Collagen InducedArthritis (CIA)

Sialoadhesin is expressed in knees of healthy mice in the bone marrow,in the thin synovial cell layer, between muscle cells and surroundingthe knee. However, its expression was found to be increased in inflamedknee joints of mice during the experimental mouse model for RA, CollagenInduced Arthritis. Especially the hyperproliferated synovium containedmany Sn⁺ cells, next to the bone marrow, between muscle cells andsurrounding the knee also seen in healthy mice.

Monoclonal Anti-Sn Antibody Conjugated with Methotrexate Detected inMouse Spleen and Mouse Knee After Injection.

200 micrograms rat monoclonal anti-Sn antibody conjugated tomethotrexate (ab-MTX) was intravenously or intraperitoneally injected inmice and detected at different time points in mouse spleen and knee bythe use of a fluorescently labeled anti-rat antibody. The injectedantibody was detected in spleen at any time point analyzed being from 30minutes until 7 days after the injection. The methotrexate coupledantibody was also able to reach the knee in a healthy mouse both via IVor IP.

Monoclonal Anti-Sn Antibody Conjugated with Methotrexate Kinetics inMouse Blood

Ab-MTX, ab or iso-MTX were detected by ELISA at time points 5, 20, 40,120 minutes, 24 u, 4 days and 7 days after injection of 200 microgramsof the corresponding antibody or conjugate in healthy mice. All threeproducts tested, showed the same kinetic profile in the blood (FIG. 14).With ab-MTX being detected at an average of 65% after 2 hours, at 30%after 24 hours, at 19% after 4 days and at 11% after 7 days comparedwith 100% set at 5 minutes after injection. Based upon these results themice were injected twice per week in the CIA experiment, but this mightbe less as the conjugate was still detected after 1 week.

Monoclonal Anti-Sn Antibody Conjugated with Methotrexate is Internalizedin Primary Mouse Macrophages

Primary alveolar mouse macrophages and CHO cells expressing mSninternalize both the Ab and the Ab-MTX when incubated at 37° C. At 4°C., only surface staining was observed.

Monoclonal Anti-Sn Antibody Conjugated with Low Dose of Methotrexate(MTX) Prevents Symptoms of Arthritis During Collagen Induced Arthritisin Mice

DBA/1 mice were immunized with collagen to initiate collagen inducedarthritis. 14 days after immunization mice were treated twice per weekwith the following conditions: PBS, high dose of MTX (35 mg/kg),negative control isotype conjugated with MTX (iso-MTX) and the anti-Snantibody conjugated with MTX (ab-MTX; equivalent of 0.2 mg/kg MTX). Allmice were clinically scored at least three times a week until 42 daysafter immunization. The high dose of MTX delayed and prevented symptomsof arthritis as expected in most mice as 33% became arthritic (FIG. 15b) with an average clinical score (FIG. 15 a) of 0.875, compared to thePBS treated group with an incidence of 58% and an average clinical scoreof 2,625. Importantly, the ab-MTX treatment prevented the clinical signsof arthritis as good, as only 3/11 (27%) mice became ill with an averagescore of 0.955, as compared to the iso-MTX group which had an incidenceof 7/11 (64%) with an average clinical score of 3,682. Longitudinalclinical scores (FIG. 15 a), determined by mixed model analysis, weresignificantly (p<0.0001) different between PBS and MTX; PBS and ab-MTX;iso-MTX and ab-MTX; iso-MTX and MTX indicating a significant effect ofthe treatment over time. However, no significant difference was foundbetween MTX and ab-MTX indicating the same anti-arthritic effects of thelow dose coupled to anti-Sn and the 175 times higher dose of solubleMTX.

Sialoadhesin Protein and Nucleotide Sequences

SEQ ID NO:5 is a protein sequence for pig sialoadhesin identified byGenBank Accession number AF509585.1. SEQ ID NO:5:

SEQ ID NO:6 is a nucleotide sequence encoding pig sialoadhesinidentified by GenBank Accession number AF509585.1. SEQ ID NO:6:

SEQ ID NO:7 is a protein sequence for mouse sialoadhesin identified byGenBank Accession number NM_(—)011426. SEQ ID NO:7

SEQ ID NO:8 is a nucleotide sequence encoding mouse sialoadhesinidentified by GenBank Accession number NM_(—)011426. SEQ ID NO:8:

SEQ ID NO:9 is a protein sequence for human sialoadhesin identified byGenBank Accession number NM_(—)023068. SEQ ID NO:9

SEQ ID NO:10 is a nucleotide sequence encoding mouse sialoadhesinidentified by GenBank Accession number NM_(—)023068. SEQ ID NO: 10:

The patents and publications mentioned herein are incorporated herein byreference. Protein and polynucleotides identified by a databaseaccession number are incorporated herein by reference in their entirety.

What is claimed is:
 1. A method of delivering methotrexate, or aprodrug, derivative, homolog or analog thereof to a cell, the methodcomprising: contacting a cell expressing sialoadhesin with a conjugate,the conjugate comprising a sialoadhesin binding moiety and methotrexate,or a prodrug, derivative, homolog or analog thereof, wherein thesialoadhesin binding moiety binds to the sialoadhesin expressed by thecell, thereby delivering methotrexate, or a prodrug, derivative, homologor analog thereof, to the cell.
 2. The method according to claim 1,wherein the cell is selected from the group consisting of a monocyte, amonocyte cell line, a macrophage, and a macrophage cell line.
 3. Themethod according to claim 1, wherein the sialoadhesin binding moiety isan antibody, or a fragment thereof, a sialoadhesin ligand or a smallmolecule.
 4. The method according to claim 1, wherein the sialoadhesinbinding moiety is a monoclonal antibody or a fragment thereof, or adomain antibody.
 5. The method according to claim 1, to treat a subjectdiagnosed as suffering from an autoimmune disease.
 6. The methodaccording to claim 1, for treating chronic inflammatory arthritidesselected from the group consisting of: rheumatoid arthritis (RA),spondyloarthritis (SpA), psoriatic arthritis (PsA), forms ofundifferentiated arthritis, and related conditions.
 7. The methodaccording to claim 6, comprising administering a therapeuticallyeffective amount of a sialoadhesin binding moiety conjugated tomethotrexate to the subject, wherein methotrexate is delivered to asialoadhesin expressing cell in the subject, thereby treating thepathological condition.
 8. The method according to claim 6, applied to amethotrexate-resistant subject.
 9. The method according to claim 1,wherein the cell is in vitro.
 10. The method according to claim 1,wherein the cell is in vivo.
 11. The method according to claim 1,wherein the cell is a mammalian cell.
 12. A method to targetmethotrexate to a sialoadhesin-expressing cell by administering to asubject a composition comprising a sialoadhesin binding moiety andmethotrexate, or a prodrug, derivative, homolog or analog thereof. 13.The method according to claim 12, wherein the sialoadhesin-expressingcell is selected from the group consisting of a monocyte, a monocytecell line, a macrophage, and a macrophage cell line.
 14. The methodaccording to claim 12, wherein the sialoadhesin binding moiety is anantibody, or a fragment thereof, a sialoadhesin ligand or a smallmolecule.
 15. A method for treating chronic inflammatory arthritidesselected from the group consisting of: rheumatoid arthritis (RA),spondyloarthritis (SpA), psoriatic arthritis (PsA), forms ofundifferentiated arthritis, and related conditions, the methodcomprising: administering a therapeutically effective amount of asialoadhesin binding moiety conjugated to methotrexate, or a prodrug,derivative, homolog or analog thereof, to the subject, and whereinmethotrexate, or a prodrug, derivative, homolog or analog thereof, isdelivered to a sialoadhesin expressing cell in the subject, therebytreating the chronic inflammatory arthritides.
 16. The method accordingto claim 15, wherein the sialoadhesin-expressing cell is selected fromthe group consisting of a monocyte, a monocyte cell line, a macrophageand a macrophage cell line.
 17. The method according to claim 15,wherein the sialoadhesin binding moiety is an antibody, or a fragmentthereof, a sialoadhesin ligand, or a small molecule.
 18. A compositioncomprising a sialoadhesin binding moiety conjugated to methotrexate, ora prodrug, derivative, homolog or analog thereof, wherein thesialoadhesin binding moiety is an antibody, or a fragment thereof, asialoadhesin ligand or a small molecule.